As formigas-cortadeiras (Hymenoptera: Formicidae), gêneros Atta e Acromyrmex, são as pragas mais importantes da silvicultura, encontradas exclusivamente nas Américas. Sua importância econômica se deve ao seu hábito alimentar de cortar material vegetal fresco – sendo algumas importantes espécies cultivadas - que serve de substrato para o fungo simbionte mutualista Leucoagaricus gongylophorus (Basidiomycota: Agaricales) o qual é cultivado em câmaras subterrâneas. As fêmeas fecundadas são responsáveis pela fundação de uma nova colônia e sofrem influência direta de microrganismos presentes no solo, assim existem evidências que fêmeas de Atta spp. recém-fecundadas evitam solos férteis, com alta carga microbiana e mesmo as áreas de matas para fundação dos seus ninhos. Os fungos entomopatogênicos e fungos antagonistas de L. gongylophorus, presentes naturalmente no solo, podem ser usados no controle biológico de formigas-cortadeiras. Os objetivos deste trabalho foram avaliar em laboratório o desenvolvendo de colônias jovens de Atta sexdens em contato com solos de área de mata e de área desnuda, realizar a bioprospecção de isolados de fungos antagonistas de L. gongylophorus (Trichoderma spp.), em solos da região de Gurupi - TO, e testar o efeito antagônico desses fungos contra L. gongylophorus. Após 106 dias da revoada, as operárias de A. sexdens das 36 colônias, logo que iniciado o experimento, removeram o solo em todos os tratamentos, e esse comportamento favoreceu a sobrevivência das colônias, apresentando uma taxa de sobrevivência de 91,67% durante as 13 semanas. Verificou-se que, após as colônias terem passado pelo período crítico de fundação, o tipo de solo parece exercer pouca influência na sobrevivência das operárias e das colônias. O solo de área de mata apresenta maior quantidade e diversidade de fungos filamentosos que o solo de área desnuda; A utilização de Tenebrio molitor como isca-viva é eficiente para bioprospecção por fungos entomopatogênicos. Todos os isolados de Trichoderma foram capazes de inibir o crescimento in vitro de L. gongylophorus; Trichoderma spirale isolado 2 apresentou crescimento micelial lento em placa de Petri e inibição contra L. gongylophorus, podendo este ser um isolado promissor em testes em campo. / The leaf-cutting ants (Hymenoptera: Formicidae), Atta and Acromyrmex genera, are the most important forest pests. They are found exclusively in Americas. Its economic importance is due to behavior to cut fresh plant material that serves as a substrate for the mutualistic symbiotic fungus Leucoagaricus gongylophorus (Basidiomycota: Agaricales) that are grown in underground chambers. Fertilized females are responsible for the founding of a new colony and they suffer direct influence of soil microorganisms. Therefore, newly fertilized females of Atta spp. should avoid fertile soils with high microbial load and even forested areas to found their nests. Entomopathogenic fungi and antagonists of L. gongylophorus are naturally present in the soil and they could be used in biological control of leaf-cutting ants. The objectives of this study were to evaluate and to compare the developing of young colonies of Atta sexdens in contact with soil from forested area and in contact with bare soils. Also, this and others procedures served to bioprospect antagonistic fungi of L. gongylophorus (Trichoderma spp.) from Gurupi region soil, and test the antagonistic effect of Trichoderma spp. against L. gongylophorus. In the first hours of start of experiments, Atta workers removed soil from colony recipients. This behavior could have favored colony survival, with a survival rate of 91,67 % during the 13 weeks. It was found that after the colonies have passed the critical period of foundation, the soil appears to exert little influence on the survival of workers and colonies. The soil from forested area had greater quantity and diversity of filamentous fungi than that from bare soil. The use of Tenebrio molitor as bait alive is efficient for bioprospecting by entomopathogenic fungi. All isolates of Trichoderma were able to inhibit in vitro growth of L. gongylophorus. Trichoderma spirale isolated 2 showed slow mycelial growth in Petri dish and strong level of inhibition against L. gongylophorus in vitro. So, this isolate is promising and should be tested in field.

Made available in DSpace on 2014-08-20T14:07:34Z (GMT). No. of bitstreams: 1 dissertacao_deise_farias_freitas.pdf: 667072 bytes, checksum: a8b684289a125aa31c5c8df5c829f81d (MD5) Previous issue date: 2010-09-09 / Leaf-cutting ants belonging to Attini (Hymenoptera-Formicidae-Myrmicinae), especially the genus Acromyrmex, are among the worst pests of Brazilian agriculture, because plants and cut pieces to carry inside their nests. The plant material is used for growing Leucoagaricus gongylophorus (Singer), from which they feed. The study aimed to determine the optimal temperature for growth and development of the symbiotic fungus Leucoagaricus gongylophorus (Singer). The fungi used in the study come from three different species of leaf cutting ants: Acromyrmex ambiguous (Emery, 1887), Acromyrmex crassispinus (Forel, 1909) and Acromyrmex heyeri (Forel, 1899), that these fungi were isolated and layered on Petri dishes containing culture medium SILVA-PINHATI et al. (2005). The treatments used were the temperatures 15, 18, 22, 25, 28 and 30ºC, which evaluated the halo of growth of the fungus 63 days after its establishment in the culture medium. The experimental design was randomized, and for each temperature were 20 repetitions. Data were analyzed by the MOBAE program. The value of the lower development threshold temperature (Tb) for the symbiotic fungus L. gongylophorus cultivated by the species A. ambiguus, were 16.22ºC, for the period studied with excellent development thermal threshold between 19.39ºC and 21.02ºC. The model adopted did not set the fungi cultivated by A. crassispinus and A. heyeri, showing that the temperature ranges adopted were not appropriate for those species, thus revealing different thermal requirements. / As formigas cortadeiras, pertencentes à tribo Attini (Hymenoptera- Formicidae-Myrmicinae,) em especial o gênero Acromyrmex, situam-se entre as piores pragas da agricultura brasileira, pois cortam plantas e transportam pedaços para interior de seus ninhos. O material vegetal é usado para o cultivo do fungo Leucoagaricus gongylophorus (Singer), do qual se alimentam. O trabalho teve como objetivo verificar a temperatura ideal para o crescimento e desenvolvimento do fungo simbionte Leucoagaricus gongylophorus. Os fungos utilizados no trabalho provêm de três diferentes espécies de formigas cortadeiras, Acromyrmex ambiguus (Emery, 1887), A. crassispinus (Forel, 1909) e A. heyeri (Forel, 1899), fungos estes que foram isolados e repicados em placas de Petri, contendo meio de cultura SILVA-PINHATI et al. (2005). Como tratamentos foram utilizados seis temperaturas (15,18, 22, 25, 28 e 30 ºC), onde se avaliou o halo de crescimento do fungo aos 63 dias após o seu estabelecimento no meio de cultura. O delineamento experimental foi inteiramente casualizado, sendo que para cada temperatura foram feitas 20 repetições. Os dados foram analisados pelo programa MOBAE. O valor do limiar térmico inferior de desenvolvimento (Tb) para o fungo simbionte L. gongylophorus cultivado pela espécie A. ambiguus, foi de 16,22ºC, para o período estudado, respectivamente, com limite térmico ótimo de desenvolvimento entre 19,39ºC e 21,02ºC. As formigas cortadeiras, pertencentes à tribo Attini (Hymenoptera- Formicidae-Myrmicinae,) em especial o gênero Acromyrmex, situam-se entre as piores pragas da agricultura brasileira, pois cortam plantas e transportam pedaços para interior de seus ninhos. O material vegetal é usado para o cultivo do fungo Leucoagaricus gongylophorus (Singer), do qual se alimentam. O trabalho teve como objetivo verificar a temperatura ideal para o crescimento e desenvolvimento do fungo simbionte Leucoagaricus gongylophorus. Os fungos utilizados no trabalho provêm de três diferentes espécies de formigas cortadeiras, Acromyrmex ambiguus (Emery, 1887), A. crassispinus (Forel, 1909) e A. heyeri (Forel, 1899), fungos estes que foram isolados e repicados em placas de Petri, contendo meio de cultura SILVA-PINHATI et al. (2005). Como tratamentos foram utilizados seis temperaturas (15,18, 22, 25, 28 e 30 ºC), onde se avaliou o halo de crescimento do fungo aos 63 dias após o seu estabelecimento no meio de cultura. O delineamento experimental foi inteiramente casualizado, sendo que para cada temperatura foram feitas 20 repetições. Os dados foram analisados pelo programa MOBAE. O valor do limiar térmico inferior de desenvolvimento (Tb) para o fungo simbionte L. gongylophorus cultivado pela espécie A. ambiguus, foi de 16,22ºC, para o período estudado, respectivamente, com limite térmico ótimo de desenvolvimento entre 19,39ºC e 21,02ºC.

Les attines sont des fourmis champignonnistes dans la zone allant des Etats-Unis à l'Argentine en passant par la Caraïbe. Les dégâts causés par ces ravageurs sont considérables et estimés à plusieurs millions de dollars par an. Elles sont de ce fait considérées comme l'un des premiers fléaux de l'agriculture de la zone (Cameron & Riggs, 1985 ; Fowler et al., 1986). En Guadeloupe, la "fourmi manioc" (Acromyrmexoctospinosus Reich) cause de nombreux dommages, aussi bien pour l'agriculture que dans les milieux naturels. Les dégâts occasionnés par ces fourmis ont nécessité l'utilisation de pesticides de synthèse. pour tenter de les combattre. La dangerosité de ces substances pour les milieux naturels et anthropisés ainsi que pour la santé publique est un contexte favorable à l'exploration de nouvelles pistes telles que l'utilisation de plantes à usages phytosanitaires. L'objet principal de cette thèse consiste à savoir si on peut déceler à partir de l'ethnopharmacologieTRAMIL des usages permettant de combattre cette fourmi et son symbiote fongique, Leucocoprinuss 'gongylophorus (Heim) MoeIler. Les protocoles retenus et présentés dans ce travail permettent d'observer les effets des usages de plantes choisis sur la survie et le comportement des insects et sur la croissance du symbiote. Ils ont été sélectionnés pour leur potentialité à mettre en évidence les différents types d'effets des extraits végétaux (insecticide par contact, insectifuge, toxique par ingestion, fongistatique ou fongicide). Six usages traditionnels issus de cinq plantes sont testés sur la fourmi: Mammea americana L., Nerium oleander L., Nicotiana tabacum L., Rollinia mucosa (Jacq.) Baill. et Trichilia pallida Sw. Cinq autres usages traditionnels sont testés sur le champignon : Allium cepa L., Allium sativum L., Lycopersicom esculentum Mill., Manihot esculenta Crantz et Senna alata (L.) Roxb. Les resultants obtenus permettent de dégager quelques pistes de lutte intéressantes contre ce ravageur. / Leaf-cutting ants are fungus-growing ants.Their geographie distribution ranges from southern United States to northern Argentina, incIuding the West Indies, Economic losses due to these ants were estimated at several million dollars per year. The latter statement makes they are considered as one of the most important pest species of the agricultural productions (Cameron & Riggs, 1985 ; Fowler et al., 1986). In (Guadeloupe, the most important invasive pest species which caused important losses in forests, gardens and field crops is 'cassava ant' (Acromyrmex octospinosus Reich). These huge losses required to use, synthetic chemical pesticides to control these leaf-cutting ants, the consequence of which is, irreversible' injuries on environment and on human health. As a matter of fact, this situation has prompted an increasing interest in alternative methods for pest control like plants with pesticide used. The main objective of this thesis is to know if traditional ethnopharmacological TRAMIL uses could help to fight against this ant and against the symbiotic fungus, Leucocoprinus gongylophorus (Heim) . Moeller. Methods used and presented in this work aIIowed to observe effects of selected plant uses on mortality and insect's behavior. The protocols performed were selected for their potentiality to detect the different types of insecticidal effects (contact toxicity, repellent effect, toxicity by ingestion, fungicidal or fungistatic). Six traditional plant uses of five plants were tested on the ant : Mammea americana L., Nerium oleander L., Nicotiana tabacum L.,Rollinia mucosa (Jacq) Baill. and Trichilia pallida Sw.Five other plants uses were tested on the fungus:Allium cepa L.,Lycopersicon esculentum Mill.,Manihot esculenta Crantz and Senna alata (L.) Roxb.The obtained résults provide us with some interesting trails to fight against this pest.

Made available in DSpace on 2014-06-11T19:28:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-02-10Bitstream added on 2014-06-13T20:37:43Z : No. of bitstreams: 1 noronhajunior_nc_me_botfca.pdf: 814511 bytes, checksum: f0ba517a59245727f01aed36f56871ba (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Empresa Privada / The objective of this paper was to have a better understanding of plant-ant-symbiotic fungus interaction focusing on foraging behavior of Atta sexdens rubropilosa workers during plant selection. Physical and chemical substrate factors were approached which can have a role on foraging material selection for symbiotic fungus cultivation besides defoliating localization in artificial plants. Tested hypothesis was that besides chemical characteristics physical stimulus and leaves localization in a plant also play a very important role in substrate selection by A. sexdens rubropilosa workers. Different shape and thickness material was used (different leaves, paper leaves with different shapes and thickness impregnated in plant extract and artificial plants). The aim was to verify the existence of physical and chemical resistance, leaf palatability and defoliating localization. Wood plants Actinostemon communis, Alchornea triplinervea, Croton floribundus, Faramea cyanea, were offered to workers and evaluated 4 according to mechanical resistance of cut and palatability. Each plant was offered individually in big disc shape (2,5cm diameter), small disc shape (0,5cm diameter) and whole leaves. Other studies were carried out for detecting physical and chemical stimulus through simulated cuts and impregnation of plant extract in paper leaves with different thickness making it possible to evaluate the combinations between physical and chemical substrate characteristics. Defoliating localization in plants was studied when offering artificial plants with four levels where either wood plant or Ligustrum sp. leaves was attached. Defoliating intensity was measured by the number of fallen leaves by ants. In order to study wood plant attractiveness small disc shaped leaves (0,5cm diameter) were offered at the same time for ants in laboratory. The end of the experiment was determined either by the carrying... (Complete abstract, click electronics address below).

Made available in DSpace on 2016-06-02T20:34:08Z (GMT). No. of bitstreams: 1 1944.pdf: 4592426 bytes, checksum: 12974a976cf746fae4024f8bf73f3159 (MD5) Previous issue date: 2007-04-25 / Universidade Federal de Sao Carlos / This work describes the biossay-guided the phytochemical study of Spiranthera odoratissima (RUTACEAE), with test against the symbiotic fungus Leucoagaricus gongylophorus, leaf-cutting ant Atta sexdens rubropilosa and pectinases enzymes of the faecal fluid of leaf-cutting ant. The phytochemical study of actives extracts allowed the identification of 15 substances: lupeol, lupenone, sitosterol, estigmasterol, campesterol, dictamine, ã-fagarine, esquimianine, 4,8-dimetoxyfuro[2,3-B]quinoline-5-7-diol, 1-methyl-2-phenyl-4- quinolone, 1-methyl-8-methoxy-2-phenyl-quinolin-4-one, limonine, limonexic acid, iso-limonexic acid e 23-hidroxy-21-oxo-dihydroodoratine. The compounds 1-methyl-8-methoxy-2-phenyl-quinolin-4-one, iso-limonexic acid e 23-hidroxy- 21-oxo-dihydroodoratine are described for the first time in the literature. 4,8- Dimetoxyfuro[2,3-B]quinoline-5-7-diol, 1-methyl-2-phenyl-4-quinolone, limonine and limonexic acid were isolated for first time from the Spiranthera genus. The branch extracts of the S. odoratissima were active against the L. gongylophorus fungus , inhibiting 100% of its growth and showed insecticide activity against leaf-cutting ants in survival biossay, with significant reduction leaf-cutting ants lifetime (5,5 days) when compared with the control group (25 days). The antimicrobial tests showed low activity of S. odoratissima against the The flavone biotransformation studies carried out with the symbiotic fungus L. gongylophorus on YNB liquid medium, solid medium and on the ants nest of the A. sexdens rubropilosa showed degradation of flavone. This degradation probably is done by the enzymatic system of the symbiotic fungus L. gongylophorus and others microorganisms presents on waste of ants nest. / Esta tese apresenta o estudo fitoquímico da planta Spiranthera odoratissima (RUTACEAE), biomonitorado por ensaios biológicos com o fungo simbionte Leucoagaricus gongylophorus, a formiga cortadeira Atta sexdens rubropilosa e as enzimas pectinases do fluido fecal da formiga cortadeira. O estudo fitoquímico dos extratos ativos levou a identificação de 15 substâncias: lupeol, lupenona, sitosterol, estigmasterol, campesterol, dictamina, ã-fagarina, esquimianina, 4,8-dimetoxifuro[2,3-B]quinolina-5-7-diol, 2-fenil-1-metilquinolin- 4-ona, 2-fenil-1-metil-8-metoxi-quinolin-4-ona, limonina, ácido limonéxico, ácido iso-limonéxico e 23-hidroxi-21-oxo-diidroodoratina. Sendo 2- fenil-1-metil-8-metoxi-quinolin-4-ona, ácido iso-limonéxico e 23-hidroxi-21- oxo-diidroodoratina inéditas na literatura e 4,8-dimetoxifuro[2,3-B]quinolina-5- 7-diol, 2-fenil-1-metil-quinolin-4-ona, limonina e ácido limonéxico isoladas pela primeira vez no gênero Spiranthera. Os extratos dos galhos da planta S. odoratissima apresentaram 100% de inibição no desenvolvimento do fungo simbionte L. gongylophorus e ação sobre a sobrevivência média de 5,5 dias em um experimento de 25 dias com as formigas cortadeiras Atta sexdens rubropilosa. Os ensaios antimicrobianos mostraram a baixa potencialidade da planta S. odoratissima contra os microrganismos testados. Os estudos de biotransformação da flavona realizados com o fungo simbionte L. gongylophorus nos meios líquidos YNB, meio sólido e no formigueiro de A. sexdens rubropilosa mostraram a possível degradação da flavona pelos sistemas enzimáticos do fungo simbionte L. gongylophorus e de outros microorganismos presentes no lixo do formigueiro.

Made available in DSpace on 2016-06-02T20:34:41Z (GMT). No. of bitstreams: 1 Retido.pdf: 19733 bytes, checksum: 6aad255badc436a06364517de2344ab6 (MD5) Previous issue date: 2013-04-24 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Leaf-cutting ants are known by their strong power of to pulling down several vegetable crops and to cause economic lost to the agriculture. Several methods have been used for their control such as the use of halogenated, sulfurous and phosphorus compounds. However, the continued use of these compounds leads to problems such as environmental contamination and then raising the needed of new strategies to control of leaf-cutting ants. In this context, this work presents a phytochemical study of the species Schinus terebinthifolius, Anacardium humile e Macairea urundeuva and the insecticide and fungicide potential of the crude extracts, fractions and isolated compounds against the leaf-cutting ants, Atta sexdens rubropilosa, and its symbiotic fungus, Leucoagaricus gongylophorus. The study of these species allowed the isolation and/or identification of several secondary metabolites including triterpens, steroids, flavonoids, biflavonoids, glycosylated flavonoids and phenolic compounds. For both species A. humile and M. urundeuva this is the first report of their chemical composition. From the biological assays with leaf-cutting ants, it was found that several extracts showed insecticidal activity as a result of the synergistic effect of their chemical composition. The elagic acid isolated from M. urundeuva showed activity against the symbiotic fungus of leaf-cutting ants. The metal complexes of flavonoid quercetin didn`t present good results on biological assays against the workers of leaf-cutting ants and its symbiotic fungus. Besides these results, this study contributed to a project that has been developed by the Natural Product Laboratory at UFSCar, which aims to evaluated the biological activity of several metal complexes with different metals and flavonoids to the control of several agriculture pests. / As formigas cortadeiras são conhecidas pelo poder de destruição de um grande número de espécies vegetais e pelo prejuízo econômico causado à agricultura. Vários métodos têm sido utilizados para o controle dos mesmos, tais como, a utilização de substâncias orgânicas halogenadas, sulfuradas e fosforadas. No entanto, a utilização contínua desses compostos tem gerado vários problemas de contaminações ambientais, sendo de extrema importância, a busca de novas estratégias de controle das formigas cortadeiras. Nesse contexto, esse trabalho apresenta o estudo fitoquímico das espécies Schinus terebinthifolius, Anacardium humile e Macairea urundeuva e avaliação do potencial inseticida e fungicida dos extratos, frações e compostos obtidos frente às formigas cortadeiras da espécie Atta sexdens rubropilosa e seu fungo simbionte Leucoagaricus gongylophorus. O estudo fitoquímico destas espécies proporcionou o isolamento e identificação de diversos metabólitos secundários, dentre eles, triterpenos, esteroides, flavonoides, biflavonoides, flavonoides glicosilados e compostos fenólicos. Para as espécies A. humile e M. urundeuva, este é o primeiro relato de suas composições químicas. A partir dos ensaios biológicos realizados com as operárias de Atta, verificou-se que vários extratos provocaram aumento na mortalidade quando comparados ao controle (dieta pura) de acordo com o teste de log-rank (p<0,05), assim, sugere-se que o potencial tóxico apresentado pelos mesmos estejam correlacionados ao efeito sinergístico. O ácido elágico e epicatequina isolados de M. urundeuva foram ativos sobre o crescimento do fungo simbionte. Os complexos metálicos com o flavonoide quercetina apresentarem resultados significativos sobre os ensaios de toxicidade por ingestão em Atta sexdens rubropilosa e seu fungo simbionte. Este trabalho contribui com um amplo projeto que vem sendo desenvolvido pelo grupo de Produtos Naturais da UFSCar, que visa avaliar a atividade inseticida e fungicida de diversos complexos de coordenação com diferentes metais e flavonoides no controle de diversas pragas.

Made available in DSpace on 2016-06-02T20:34:56Z (GMT). No. of bitstreams: 1 6246.pdf: 4525981 bytes, checksum: 69b0012bfc897e466c42b6036376948c (MD5) Previous issue date: 2014-08-12 / Universidade Federal de Minas Gerais / Polygalacturonases (PGases) are enzymes which hydrolyze glycosidic linkages &#61537;- 1,4 between pectic acid residues, and are produced by plants, fungi, bacteria and yeasts. The fungus L. gongylophorus, Atta sexdens ant mutualist, secretes enzymes with PGase activity. The project was developed in two approaches: study with recombinant and native PGase. The activity on polygalacturonic acid of native PGase in fungus culture medium was determined, and its purification from crude extract was performed by (NH4)2SO4 precipitation and molecular exclusion chromatography. PGase kinetic parameters (Vmax and KM), optimal pH and temperature were determined, and the enzyme was immobilized on magnetic particles, proved to be a good method to future work inhibitors search. A cDNA library was constructed from total RNA obtained from L. gongylophorus culture in induction medium. 816 clones from the library were sequenced, allowing the identification of enzymes sequences involved in the plant cell wall degradation, some already in cloning and expression in our laboratory. Using a deposited L. gongylophorus PGase (PGase-Lg) sequence (GenBank: ADV30326.1), primers were designed to amplify the PGase-Lg gene, with cleavage sites for EcoRI, HindIII and NotI enzymes, respecting the pETSUMO (for E. coli expression) and pPICZ&#61537;A (for P.pastoris expression) reading phase vectors. From the cDNA, the PGase-Lg ORF encoding was amplified by PCR and cloned in both vectors. The clones were confirmed by plasmid DNA extraction and PCR. E. coli expression experiments of pETSUMO-PGase-Lg construction showed a great expression of His.tag-SUMO.tag-Lg-PGase fusion protein in the insoluble form and many expression and solubility assays were inefficient in solubility of expressed fusion protein. The protein refolding was performed and this was obtained in soluble form but lacks PGase activity, showing that folding may not have been correctly or E. coli fusion protein expressed does not undergo post-translational modifications to have enzymatic activity. The rPGase-Lg expressed in P. pastoris showed PGase activity on polygalacturonic acid and electrophoresis analysis suggest more than one enzyme expression, with different glycosylation content. / Poligalacturonases (PGases) são enzimas que hidrolisam ligações glicosídicas &#61537;-1,4 entre resíduos de ácido péctico, e são produzidas por plantas, fungos, bactérias e leveduras. O fungo L. gongylophorus mutualista da formiga Atta sexdens, secreta enzimas com atividade PGase. O projeto foi desenvolvido sob duas abordagens: estudo com a PGase nativa e recombinante. A atividade sobre ácido poligalacturônico da PGase nativa foi determinada no extrato bruto do fungo, e sua purificação foi feita por precipitação com (NH4)2SO4 e cromatografia de exclusão molecular. Temperatura e pH ótimos e parâmetros cinéticos Vmax e KM foram determinados, e a PGase foi imobilizada em partículas magnéticas, mostrando ser um bom método na busca de inibidores. Construiu-se uma biblioteca de cDNA a partir de RNA total obtido de cultura de L. gongylophorus em meio indutor. Foram sequenciados 816 clones da biblioteca, permitindo identificar sequências de enzimas envolvidas na degradação da parede celular vegetal, algumas em fase de clonagem e expressão em nosso laboratório. Utilizando uma sequência de PGase de L. gongylophorus (PGase-Lg) depositada (GenBank: ADV30326.1), foram desenhados oligonucleotídeos para a amplificação do gene da PGase-Lg, com sítios de clivagens das enzimas EcoRI, HindIII e NotI, respeitando a fase de leitura dos vetores pETSUMO (para expressão em E.coli) e pPICZ&#61537;A (para expressão em P.pastoris). A partir do cDNA, a ORF codificante da PGase-Lg foi amplificada por PCR e clonada nos dois vetores. Os clones foram confirmados por extração de DNA plasmidial e PCR. Experimentos de expressão em E. coli da construção pETSUMO-PGase-Lg apresentaram grande expressão da proteína em fusão His.tag-SUMO.tag-PGase-Lg na forma insolúvel, e vários ensaios de expressão e solubilidade se mostraram ineficientes na solubilidade da proteína em fusão expressa. Realizou-se o refolding da proteína e esta foi obtida na forma solúvel, porém sem atividade, mostrando que o enovelamento pode não ter ocorrido corretamente ou que a proteína em fusão expressa pela E. coli não sofre as modificações pós-traducionais necessárias. A rPGase-Lg expressa em P. pastoris apresentou atividade PGase em ácido poligalacturônico, e análise em eletroforese sugere a expressão de mais de uma proteína, com conteúdos diferentes de glicosilação.