Orientador: Eleni Gomes / Resumo: O grande acumulo de biomassa lignocelulósica oriunda da produção de etanol no Brasil contribui para um aumento em pesquisas para utilização para etanol de segunda geração e produção de enzimas com maior valor agregado. Considerando que o grupo de pesquisa ao qual esse trabalho está vinculado tem desenvolvido pesquisas com celulases, hemicelulases, ligninases, pectinases, esse estudo teve por objetivo selecionar leveduras produtoras dessas enzimas, realizar a caracterização físico-química e a purificação enzimática. Diversos trabalhos tem sido desenvolvidos a fim do reaproveitamento de material lignocelulósico oriundo da produção de etanol por cana-de-açúcar para a produção de enzimas de interesse comercial e sua principal aplicação na produção de etanol de segunda geração. Foi selecionado duas leveduras entre 30 escolhidas da coleção do Laboratório de Bioquímica e Microbiologia Aplicadas UNESP/IBILCE: A. pullulans LB 3.1 e A. leucospermi LB 86 e que foram cultivadas com farelo de trigo. Ambas foram capazes de produzir xilanase, β-xilosidase e β-glicosidase, sendo a maior atividade de xilanase de 72 U ml-1 para LB 3.1 de 5,5 U ml-1 para LB 86, bem como a atividade de β-xilosidase que foi (6,7 U ml-1 ) para LB 3.1 e (4,0 U ml-1 ). Com relação à influência do pH na atividade enzimática foi observado perfis similares para xilanase e βxilosidase produzidas pela levedura A. pullulans LB 3.1, com uma maior atividade na faixa de pH 2,5 a 3,5. Essa característica foi observada também ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The large accumulation of lignocellulosic biomass from ethanol production in Brazil contributes to an increase in research for use for second generation ethanol and the production of enzymes with greater added value. Considering that the research group to which this work is linked has developed research with cellulases, hemicellulases, ligninases, pectinases, this study aimed to select yeasts that produce these enzymes, perform physical-chemical characterization and enzymatic purification. Several works have been developed in order to reuse lignocellulosic material from the production of ethanol by sugarcane for the production of enzymes of commercial interest and its main application in the production of second generation ethanol. Two yeasts were selected from 30 chosen from the collection of the Laboratory of Applied Biochemistry and Microbiology UNESP / IBILCE: A. pullulans LB 3.1 and A. leucospermi LB 86 and which were grown with wheat bran. Both were able to produce xylanase, β-xylosidase and β-glycosidase, with the highest xylanase activity being 72 U ml-1 for LB 3.1 and 5.5 U ml-1 for LB 86, as well as the activity of β-xylidasidase which was (6.7 U ml-1) for LB 3.1 and (4.0 U ml-1). Regarding the influence of pH on enzyme activity, similar profiles were observed for xylanase and β-xylosidase produced by the yeast A. pullulans LB 3.1, with a greater activity in the pH range 2.5 to 3.5. This characteristic was also observed for the βxylosidase of A. leucospermi LB 86, w... (Complete abstract click electronic access below) / Doutor

O objetivo desse trabalho foi estudar o potencial de xilanases presentes no filtrado de cultura e de xilanases purificadas de Penicillium chrysogenum no processo de branqueamento de polpa celulósica de pseudocaule de bananeiras frutíferas. Inicialmente, estabeleceu-se o meio e o tempo de cultivo ótimos para a produção de xilanase pelas linhagens IFO-4626 e M-85 de Penicillium chrysogenum. Ambas as linhagens apresentam, além da alta atividade de xilanase, alta atividade de pectinases e baixa atividade de celulases, características que contribuem para seu emprego no processamento de polpa e(ou) fibras celulósicas. Para a linhagem IFO-4626, a melhor produção de xilanase foi obtida no meio Ferreira após 60h de cultivo, usando como indutor xilana de oat spelt. O desempenho da linhagem M-85 só se iguala ao da IFO-4626 no tempo 72h, no meio Haas. Como fontes de carbono, palha de cana-de-açúcar e fibra de coco podem substituir a xilana de oat spelt na produção de xilanase pela linhagem IFO-4626. Xilose pode induzir a síntese de xilanase quando nenhuma fonte de xilana é adicionada ao meio. A inibição da atividade de xilanase foi observada nos meios com xilana e glicose (1%) ou galactose (1%). Nos experimentos de purificação da xilanase foi usado o filtrado de cultura obtido após 72h de cultivo da linhagem IFO-4626 no meio Ferreira. Dois picos com atividade de xilanase foram obtidos após a eluição em uma coluna de troca aniônica DEAE-Sephacel. A xilanase I, com massa molecular de 12,6 kDa, Km = 12,14 mg/mL e Vmáx = 7,75 U/µg de proteína, não se ligou à resina e a xilanase II, com massa molecular de 20 kDa, Km = 39,32 mg/mL e Vmáx = 1579,62 mg/mL, foi eluída com 100 mM de NaCl. Tanto as xilanases presentes no filtrado de cultura como as xilanases I e II, apresentaram boa estabilidade térmica a 40°C e 50°C e em valores de pH de 3,5 a 9,0. Porém, além de não possuírem boa estabilidade a 60°C, suas temperaturas e pH ótimos de reação são baixos. As xilanases presentes no filtrado de cultura foram ativadas pela presença de Fe+2, Mn+2, Ca+2 e ditiotreitol (DTT) e inibidas pela presença de Zn+2, dodecil sulfato de sódio (SDS), Pb+2 e Hg+2. A atividade da xilanase I foi estimulada por DTT, Ca+2 e Mn+2 e inibidas por Cu+2, Zn+2, SDS e Hg+2. Já a xilanase II foi inibida por Hg+2 e ativada por diversos íons: Mn+2, Co+2, Fe+2, Ba+2, Ca+2, Cu+2, Mg+2 e por NH4+ e DTT. As xilanases presentes no filtrado de cultura da linhagem IFO-4626 e as xilanases purificadas I e II favoreceram a liberação de cromóforos a 237nm e de açúcares redutores. Porém, as enzimas presentes no filtrado de cultura mostraram-se mais adequadas para liberação de cromóforos com absorção em diferentes comprimentos de onda. Ou seja, constatou-se que as enzimas presentes no filtrado de cultura possuem melhor potencial do que as xilanases purificadas I e II, para favorecer o posterior branqueamento da polpa kraft de pseudocaules de bananeiras frutíferas. / The aim of this work was to study the potential of xylanases present in culture filtrate of Penicillium chrysogenum and of this xylanases purified in favor of the pre-bleaching of pseudo-stem cellulosic pulp of banana trees. Early, it was established the optimal media and time of culture to production of xylanases by IFO-4626 and M-85 Penicillium chrysogenum strains. Both strains have presented such characteristics as to contribute to their use in pulp and/or cellulosic fibers industrial process, besides the high activity of pectinases and the low activity of cellulases. To the IFO-4626 strain, the best production of xylanases using as inductor oat spelt xylan was obtained in the Ferreira medium, after 60h of culture. The performance of M-85 strain only was equal to that at the 72 hours in the Haas medium. As carbon sources, both sugar-cane straw and coconut fiber can substitute the oat spelt xylan in the production of xylanase by IFO-4626 strain. Xylose can induce the synthesis of xylanase when no xylan source was added to the culture medium. The inhibition of activity of xilanase was observed in medium with xylan plus glycose (1%) or galactose (1%). In the essays about xylanase purification, it was used the culture filtrate of IFO-4626 strain, obtained after 72 hours of culture in Ferreira medium. Two peaks with xylanase activity were obtained after the elution in an anionic-exchange column DEAE-Sephacel. The xylanase I, showed molecular mass of 12.6 kDa, Km = 12.14mg/mL and Vmax = 7.75 U/µg of protein, and it was not bind to the resin, and the xylanase II, with molecular mass of 20 kDa, Km = 39.32 mg/mL and Vmáx = 1579.62 mg/mL, was eluted with NaCl 100 mM. The xylanases in the culture filtrate, and the xylanases I and II, have presented good stability at 40°C and 50°C, and in pH values 3.5 to 9.0, despite of having no good stability at 60°C, their optimal temperatures and pH of reaction are low. The xylanases present in culture filtrate were activated by the presence of Fe+2, Mn+2, Ca+2 and dithiothreitol (DTT) and inhibited by the presence of Zn+2, sodium dodecyl sulphate (SDS), Pb+2 and Hg+2. The xylanase I activity was stimulated by DTT, Ca+2, and Mn+2, and inhibited by Cu+2, Zn+2, SDS, and Hg+2. On the other hand, the xylanase II was inibited by Hg+2, and it was activated by several ions, like as: Mn+2, Co+2, Fe+2, Ba+2, Ca+2, Cu+2, Mg+2, and by NH4+ and DTT. The xylanases from IFO-4626 strain, present in the culture filtrate, and the purified xylanases I and II favored the release of chromophores at 237 nm, and release of reducing sugars. Although, the enzymes present in the culture filtrate show better adequacy than the purified ones in order to release chromophores, they show absorption in different wave lengths. In other words, it was verified that the enzymes present in the culture filtrate have the best potential to favor the further bleaching of the banana tree pseudo-stem kraft pulp.

O objetivo desse trabalho foi estudar o potencial de xilanases presentes no filtrado de cultura e de xilanases purificadas de Penicillium chrysogenum no processo de branqueamento de polpa celulósica de pseudocaule de bananeiras frutíferas. Inicialmente, estabeleceu-se o meio e o tempo de cultivo ótimos para a produção de xilanase pelas linhagens IFO-4626 e M-85 de Penicillium chrysogenum. Ambas as linhagens apresentam, além da alta atividade de xilanase, alta atividade de pectinases e baixa atividade de celulases, características que contribuem para seu emprego no processamento de polpa e(ou) fibras celulósicas. Para a linhagem IFO-4626, a melhor produção de xilanase foi obtida no meio Ferreira após 60h de cultivo, usando como indutor xilana de oat spelt. O desempenho da linhagem M-85 só se iguala ao da IFO-4626 no tempo 72h, no meio Haas. Como fontes de carbono, palha de cana-de-açúcar e fibra de coco podem substituir a xilana de oat spelt na produção de xilanase pela linhagem IFO-4626. Xilose pode induzir a síntese de xilanase quando nenhuma fonte de xilana é adicionada ao meio. A inibição da atividade de xilanase foi observada nos meios com xilana e glicose (1%) ou galactose (1%). Nos experimentos de purificação da xilanase foi usado o filtrado de cultura obtido após 72h de cultivo da linhagem IFO-4626 no meio Ferreira. Dois picos com atividade de xilanase foram obtidos após a eluição em uma coluna de troca aniônica DEAE-Sephacel. A xilanase I, com massa molecular de 12,6 kDa, Km = 12,14 mg/mL e Vmáx = 7,75 U/µg de proteína, não se ligou à resina e a xilanase II, com massa molecular de 20 kDa, Km = 39,32 mg/mL e Vmáx = 1579,62 mg/mL, foi eluída com 100 mM de NaCl. Tanto as xilanases presentes no filtrado de cultura como as xilanases I e II, apresentaram boa estabilidade térmica a 40°C e 50°C e em valores de pH de 3,5 a 9,0. Porém, além de não possuírem boa estabilidade a 60°C, suas temperaturas e pH ótimos de reação são baixos. As xilanases presentes no filtrado de cultura foram ativadas pela presença de Fe+2, Mn+2, Ca+2 e ditiotreitol (DTT) e inibidas pela presença de Zn+2, dodecil sulfato de sódio (SDS), Pb+2 e Hg+2. A atividade da xilanase I foi estimulada por DTT, Ca+2 e Mn+2 e inibidas por Cu+2, Zn+2, SDS e Hg+2. Já a xilanase II foi inibida por Hg+2 e ativada por diversos íons: Mn+2, Co+2, Fe+2, Ba+2, Ca+2, Cu+2, Mg+2 e por NH4+ e DTT. As xilanases presentes no filtrado de cultura da linhagem IFO-4626 e as xilanases purificadas I e II favoreceram a liberação de cromóforos a 237nm e de açúcares redutores. Porém, as enzimas presentes no filtrado de cultura mostraram-se mais adequadas para liberação de cromóforos com absorção em diferentes comprimentos de onda. Ou seja, constatou-se que as enzimas presentes no filtrado de cultura possuem melhor potencial do que as xilanases purificadas I e II, para favorecer o posterior branqueamento da polpa kraft de pseudocaules de bananeiras frutíferas. / The aim of this work was to study the potential of xylanases present in culture filtrate of Penicillium chrysogenum and of this xylanases purified in favor of the pre-bleaching of pseudo-stem cellulosic pulp of banana trees. Early, it was established the optimal media and time of culture to production of xylanases by IFO-4626 and M-85 Penicillium chrysogenum strains. Both strains have presented such characteristics as to contribute to their use in pulp and/or cellulosic fibers industrial process, besides the high activity of pectinases and the low activity of cellulases. To the IFO-4626 strain, the best production of xylanases using as inductor oat spelt xylan was obtained in the Ferreira medium, after 60h of culture. The performance of M-85 strain only was equal to that at the 72 hours in the Haas medium. As carbon sources, both sugar-cane straw and coconut fiber can substitute the oat spelt xylan in the production of xylanase by IFO-4626 strain. Xylose can induce the synthesis of xylanase when no xylan source was added to the culture medium. The inhibition of activity of xilanase was observed in medium with xylan plus glycose (1%) or galactose (1%). In the essays about xylanase purification, it was used the culture filtrate of IFO-4626 strain, obtained after 72 hours of culture in Ferreira medium. Two peaks with xylanase activity were obtained after the elution in an anionic-exchange column DEAE-Sephacel. The xylanase I, showed molecular mass of 12.6 kDa, Km = 12.14mg/mL and Vmax = 7.75 U/µg of protein, and it was not bind to the resin, and the xylanase II, with molecular mass of 20 kDa, Km = 39.32 mg/mL and Vmáx = 1579.62 mg/mL, was eluted with NaCl 100 mM. The xylanases in the culture filtrate, and the xylanases I and II, have presented good stability at 40°C and 50°C, and in pH values 3.5 to 9.0, despite of having no good stability at 60°C, their optimal temperatures and pH of reaction are low. The xylanases present in culture filtrate were activated by the presence of Fe+2, Mn+2, Ca+2 and dithiothreitol (DTT) and inhibited by the presence of Zn+2, sodium dodecyl sulphate (SDS), Pb+2 and Hg+2. The xylanase I activity was stimulated by DTT, Ca+2, and Mn+2, and inhibited by Cu+2, Zn+2, SDS, and Hg+2. On the other hand, the xylanase II was inibited by Hg+2, and it was activated by several ions, like as: Mn+2, Co+2, Fe+2, Ba+2, Ca+2, Cu+2, Mg+2, and by NH4+ and DTT. The xylanases from IFO-4626 strain, present in the culture filtrate, and the purified xylanases I and II favored the release of chromophores at 237 nm, and release of reducing sugars. Although, the enzymes present in the culture filtrate show better adequacy than the purified ones in order to release chromophores, they show absorption in different wave lengths. In other words, it was verified that the enzymes present in the culture filtrate have the best potential to favor the further bleaching of the banana tree pseudo-stem kraft pulp.

&alpha / -Galactosidase is an exo-glycosidase that hydrolyses non-reducing, &alpha / -1,6-linked &alpha / -galactose units from oligosaccharides, galactomannans, and galactolipids. &alpha / -Galactosidase activity has biotechnological, industrial, and medical importance. &alpha / -Galactosidase from A. fumigatus IMI 385708, in particular, can catalyse unique hydrolysis and transgalactosylation reactions on polymeric substrates. In this study, &alpha / -galactosidase of the human pathogen A. fumigatus IMI 385708 was first produced in a GRAS organism, Aspergillus sojae. For this aim, &alpha / -galactosidase gene (aglB) of A. fumigatus IMI 385708 was ligated onto pAN52-4 vector (Acc. No: Z32699) and transformed into Aspergillus sojae ATCC11906, under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (gpdA) of A. nidulans and the signal sequence of glucoamylase gene (glaA) of A. niger. This allowed high level of &alpha / -galactosidase production on glucose instead of locust bean gum (2.45 U/mL), corresponding to a 3-fold increase in volumetric production. Next, using response surface methodology, carbon and nitrogen sources and agitation speed were optimized (10.5% molasses (w/v) / 1.3% NH4NO3 (w/v) / 276 rpm). Compared to non-optimized cultivation, a further 4-fold increase in &alpha / -galactosidase production (10.4 U/mL) was achieved. Recombinant &alpha / -galactosidase was purified 18.7-fold using Anion Exchange and Hydrophobic Interaction Chromatography with an overall yield of 56% and 64.7 U/mg protein. The Vmax and Km values for the hydrolysis of p-nitrophenyl &alpha / -D-galactopyranoside were 78 U/mg protein and 0.45 mM, respectively. Optimum pH and temperature for &alpha / -galactosidase activity were between pH 4&ndash / 6 and 50&ndash / 60 &deg / C, respectively. Among the tested chemical agents, Ag+, Hg2+, and Fe2+ drastically decreased the activity, while biotin, I+1, Mn+2, Pb+2, Li+1, and Mg+2 enhanced between 12&ndash / 29%. To analyse the influence of osmotic stress as a means of further inducing &alpha / -galactosidase production, salt was added into the complete growth medium. In addition to enzyme production, fungal growth and morphology were analysed for both &lsquo / salt-adapted&rsquo / and &lsquo / salt non-adapted&rsquo / A. sojae Ta1 cells in the presence of KCl, MgCl2, MgSO4, NaCl, and Na2SO4 at 1 M and 2 M. Accordingly, 3-fold increase in &alpha / -galactosidase production was achieved by non-adapted cells in the presence of 1 M NaCl. Exposure of A. sojae Ta1 cells to salt resulted in predominantly mycelial form, rather than the pellet form observed under normal conditions. Finally, the transgalactosylation ability of &alpha / -galactosidase was studied. &alpha / -Galactosidase efficiently catalysed galactose transfer to different monosaccharides and disaccharides in the presence of pNP&alpha / Gal as monitored by TLC, ESI-MS, and HPLC.

Du fait de l’apparition de microorganismes pathogènes ayant une résistance aux antibiotiques actuels, la recherche de nouvelles molécules bioactives possédant une application médicale est devenue une préoccupation mondiale. Saccharothrix algeriensis, une bactérie filamenteuse de l’ordre des actinomycètes a montré une étonnante capacité à produire des molécules bioactives qui appartiennent aux dithiolopyrrolones, ayant de remarquables propriétés à la fois antibiotiques et anticancéreuses. Lors de ce projet de thèse, l’identification du cluster de gènes de la voie de biosynthèse des dithiolopyrrolones chez Sa. algeriensis est envisagée. Suite au séquençage du génome de Sa. algeriensis, une approche génomique ou « genome mining » est suivie, cette approche a révélé un cluster thi potentiellement responsable de la voie de biosynthèse des dithiolopyrrolones chez Sa. algeriensis. Ce cluster contient 12 gènes, dont 8 gènes de biosynthèse, 3 gènes régulateurs et un gène transporteur. Les analyses in silico des gènes ont montré que la cystéine est le substrat d’une NRPS. Les analyses transcriptionelles ont montré que les trois gènes clés codent pour une NRPS, une thiorédoxine et une thioestérase qui pourraient être impliquées dans la biosynthèse des dithiolopyrrolones. Deux gènes actA et actB codant pour des acyltransférases putatives ont été identifiés. Les analyses transcriptionelles suggèrent qu’actA et actB pourraient être responsables de l’acylation de la pyrrothine. Finalement, la caractérisation de deux activités enzymatiques, acétyltransférase et benzoyltransférase, présentes dans l’extrait brut de Sa. algeriensis, ont permis de déterminer les paramètres optimaux (pH et T °C) de la réaction enzymatique. Enfin, les paramètres cinétiques de ces activités ont des valeurs complètements différentes, ce qui confirme la présence d’au moins deux activités différentes chez Sa. algeriensis. / Due to the emergence in the last decades of new and old infectious diseases to existing antibiotics, the research for new bioactive molecules which possess medical applications become a global occupation. Saccharothrix algeriensis, filamentous bacteria of actinomycetes order showed a surprising ability to produce bioactive molecules belongs to dithiolopyrrolones with remarkable properties of both antibiotics and anticancer. In this thesis, the identification of dithiolopyrrolones biosynthetic gene cluster in Sa. algeriensis was investigated. Through S. algeriensis genome sequencing, a genomics approach "genome mining" was followed, this approach has revealed a potentially thi cluster responsible for dithiolopyrrolones biosynthesis pathway in Sa algeriensis. This cluster contains 12 genes, including 8 biosynthesis genes, three regulatory genes and one transporter gene. The in silico analysis of this cluster showed that the cysteine is the substrate of the NRPS. The transcriptional analyzes showed that the three key genes which encode for NRPS, thioredoxin and thioesterase could be involved in dithiolopyrrolone biosyntheses. Two genes, actA and actB, encode for two putative acyltransferases were identified, the transcriptional analyzes suggests that these genes may be responsible for the acylation of pyrrothine core. The characterizations of two activities, acetyltransferase and benzoyltransferase, in the crude extract of Sa. algeriensis led the determination of the optimal parameters (pH and T °C) to detect these activities. Moreover, the effect of temperature and pH on these activities was determined. Finally, the kinetic parameters of these activities showed different values, which confirm the presence of, at least, two activities in Sa. algeriensis.

Made available in DSpace on 2016-06-02T20:36:48Z (GMT). No. of bitstreams: 1 Retido.pdf: 19733 bytes, checksum: 6aad255badc436a06364517de2344ab6 (MD5) Previous issue date: 2013-08-30 / Universidade Federal de Minas Gerais / Xylanases are enzymes which randomly cleave the main chain of xylan, the most abundant non-cellulosic polysaccharide of plants cell wall. Xylanases are commonly produced by a wide range of organisms including bacteria, algae, fungi, protozoa, and certain herbivorous insects and crustaceans also produce xylanases. Leucoagaricus gongylophorus, a mutualistic fungus of leafcutting ant Atta sexdens, secretes enzymes with xylanolytic activity and the gene encoding a xylanase was recently identified. In this work the xylanolytic profile of L. gongylophorus was studied and two enzymes with xylanolytic activity (XyLg1 and XyLg2) were isolated, purified and characterized. XyLg1 has a molecular mass of about 38kDa and pI greater than 4.8. For beechwood xylan substrate XyLg1 showed optimum temperature of 40 °C, optimum pH between 8.5 to 10.5 and Km =14, 7 ± 7.6 mg.ml-1. Due to these features XyLg1 may be used in processes such as bio-bleaching pulp. XyLg1 was also analyzed by mass spectrometry technique being associated with a polygalacturonase of the same fungus. Kinetic studies of the XyLg1 using polygalacturonic acid as substrate were developed (optimum pH= 5.5, optimum temperature between 50 and 60 ° C and Km= 2.2 ± 0.5 mg.ml-1). XyLg2 has molecular weight of about 24kDa and pI less than 4.8, and thus it is an acid protein. Parameter such as optimum temperature (70 °C) and pH (4.0) as well as the kinetic parameters (Km 7.4 ± 2.0 mg.ml-1) using beechwood xylan as substrate were determined for XyLg2. This enzyme exhibits desirable characteristics for improving animal feed, for example. LgXyn2 shows no activity with polygalacturonic acid. For the purpose of producing larger amount of xylanase from the L. gongylophorus the gene sequence encoding a xylanase (LgXyn1, GenBank: EF208066.1) was used to synthesize forward and reverse primers and was possible to amplify a different gene (xyl) that encodes the synthesis of a new xylanase called here LgXyn2. The gene was cloned into pETSUMO vector and the recombinant expressed in E.coli has no activity even when histidine tail (fusion) is removed with Sumo protease. These results suggest that the glycosylation is an important factor for xylanolitic activity. Then the xyl gene was cloned into pPICZalphaA vector and LgXyn2 was expressed in P. pastoris, secreted into the extracellular medium and the enzyme has xylanolitic activity. This result showed that the new gene (xyl) encodes a functional enzyme and that P. pastoris is a efficient system to obtain the active enzyme. / Xilanases são enzimas que, randomicamente, clivam a cadeia principal da xilana, o polissacarídeo não celulósico mais abundante da parede celular de plantas. As xilanases são comumente produzidas por uma grande gama de organismo incluindo bactérias, algas, fungos, protozoários, sendo que alguns insetos herbívoros e crustáceos também produzem xilanases. Leucoagaricus gongylophorus, é um fungo mutualístico da formiga saúva Atta sexdens, que secreta enzimas com atividade xilanolítica e o gene que codifica para uma xilanase foi recentemente identificado. Neste trabalho o perfil xilanolítico do fungo L. gongylophorus foi estudado e duas enzimas nativas com atividade xilanolítica (XyLg1 e XyLg2) foram isoladas, purificadas e caracterizadas. XyLg1 apresenta massa molecular aproximado de 38kDa e pI maior do que 4,8. Para o substrato xilana de faia a enzima apresentou temperatura ótima de 40°C, pH ótimo entre 8,5 a 10,5 e Km14,7 ± 7,6 mg.ml- 1. Devido a essas características a XyLg1 poderá ser utilizada em processos como o biobranqueamento da celulose. XyLg1 também foi analisada por espectrometria de massas sendo relacionada com uma poligalacturonase do mesmo fungo. Estudos cinéticos da XyLg1 utilizando ácido poligalacturônico como substrato foram realizados (pHótimo= 5,5, temperatura ótima entre 50 e 60°C, Km 2,2 ± 0,5 mg.ml-1). A XyLg2 apresenta massa molecular aproximada de 24kDa e pI menor que 4,8, sendo assim uma proteína ácida. Parâmetros ótimos de temperatura (70°C) e pH (4,0), assim como o parâmetro cinético (Km 7,4 ± 2,0 mg.ml-1) utilizando xilana de faia como substrato foram determinados para a XyLg2. Esta enzima apresenta características desejáveis para melhoramento da alimentação animal, por exemplo. A LgXyn2 não apresenta atividade frente ao ácido poligalacturônico. Com o propósito de produzir elevados níveis de xilanases do L. gongylophorus, a sequência que codifica para a xilanase (LgXyn1, GenBank: EF208066.1) foi utilizada para a síntese de oligonucleotídeos foward e reverse e foi possível amplificar um gene diferente (xyl) que codifica a síntese de uma nova xilanase denominada aqui LgXyn2. O gene foi clonado no vetor pETSUMO e a enzima recombinante expressa em E. coli não apresenta atividade mesmo quando a cauda de histidina (fusão) é removida com Sumo protease. Então o gene xyl foi clonado no vetor pPICZα-A e a LgXyn2 foi expressa em P. pastoris, secretada para o meio extracelular e a enzima apresenta atividade xilanolítica. Este resultado mostra que o gene (xyl) codifica para uma enzima funcional e que a P. pastoris é um sistema eficiente para obter esta xilanase.