Intensive and semi-intensive aquaculture systems are dependent on nutrient input either in the form of supplemental or complete feeds. Most complete diets still include high fish meal (FM) levels (≥10%). However, as the industry attempts to reduce its reliance on FM, feeds must now be formulated with much lower levels especially for omnivorous species such as tilapia. By 2015, mean FM inclusion in tilapia diets was projected to fall below 3% and be further reduced to 1% by 2020. In the global context of competition for crops, finding suitable plant-based replacers for FM and meeting the increasing demand for seafood, lower-cost and under-utilised plant feedstuffs are now receiving greater attention. The study was divided into three distinct components – field survey, growth experiments, and life cycle assessment. Field surveys were used to contextualise the growth experiments and assess commercialisation opportunities for multi-enzyme inclusion in tilapia feeds. Two sets of digestibility and growth experiments were designed to evaluate the feasibility of using high inclusions of plant-based ingredients sourced from locally available feedstuffs in Thailand to substitute FM at low inclusion levels (0 – 5%). The research evaluated the hypothesis regarding the potential of exogenous enzymes (protease, xylanase and phytase) to minimize anti-nutritional effects on nutrient digestibility of proteins, polysaccharides and phosphorus in tilapia. The research also assessed the secondary effects of enzyme supplementation on economic efficiency and life cycle environmental impacts. Tilapia is the second most cultured finfish globally and Thailand is the sixth largest producer. Based on the findings of the field survey, feeding practices of Thai tilapia farmers were confirmed to be diverse. Feed inputs included, but were not limited to, agro-industrial by-products (e.g. rice bran, corn bran etc.) and commercial diets. Commercial diets contained 15 – 30% crude protein and lower protein livestock diets (i.e. pig ration) were often used for supplemental feeding or “fattening”. The experimental low FM diets were therefore formulated as grow-out or “fattening” diets for semi-intensive green-water systems, a prominent feature (>60%) of Thai tilapia farming. In Phase 1, the digestibility experiment assessed the digestibility and growth in tilapia fed 0%, 3% and 5% FM diets with and without xylanase (0.385 g kg-1) and phytase (0.075 g kg-1). Performance decreased significantly with declining FM levels. No differences in feed intake, feed conversion ratio (FCR), specific growth rate (SGR) and weight gain were observed between the enzyme and control diets. Nevertheless, tilapia fed the enzyme supplemented 3% FM and control 5% FM performed similarly (P < 0.05). No enzyme-related effects were noticed for protein digestibility but phosphorus (P) digestibility improved by 9%, except at 0% FM level (P > 0.05). The enzymes had no apparent influence on nitrogen (N) retention contrary to previous studies, however, higher retention for P was observed. Villus length decreased with declining FM levels yet no improvements were seen in tilapia fed enzyme diets. In a simultaneous grow-out experiment, the six experimental diets were compared to an industry 10% FM standard. Conversion ratio was the lowest (1.66) in adult tilapia fed 10% FM diet however the enzyme supplemented 0% FM fed fish had a comparatively low FCR of 1.67. There were no significant enzyme-related effects on weight gain¸ SGR and protein efficiency. Proximal villi results were inconsistent. The cost of feed decreased with declining FM levels but increased with enzyme inclusion. Nevertheless, the economic returns per kg of whole fish produced were better using enzyme supplemented diets compared to the controls. Though the size of the effects on growth and nutrient utilisation were modest, the findings suggested that xylanase and phytase had some level of synergistic action on the targeted anti-nutrients. However, further research was required. In Phase 2, two control diets (2% FM, negative control (NC) and 10% FM, positive control (PC)) were compared with three enzyme supplemented 2% FM diets (NO-PRO, 0.385 g kg-1 xylanase and 0.075 g kg-1 phytase only; LO-PRO, xylanase + phytase + 0.2 g kg-1 protease and HI-PRO, xylanase + phytase + 0.4 g kg-1 PRO). Growth performances improved with enzyme supplementation compared to the NC (P < 0.05). Of the enzyme supplemented diets, the LO-PRO diet showed the highest improvements in weight gain (26%) and feed intake (19%), the latter comparing statistically to the 10% FM PC diet. The HI-PRO diet had the best FCR (1.88), again comparable to the PC (1.73). The NO-PRO diet had the highest protein, P, lipid and energy digestibility, suggesting no additive effect of protease on these coefficients. In terms of gut histomorphology, the LO-PRO and PC diets had the highest measurements and were statistically similar which may have explained similarities in feed intake. Compared to the NC, the HI-PRO diet produced the highest level of change in net profit due to gains in feeding efficiency however, the LO-PRO showed better improvements in terms of growth. Based on these findings, the ternary combination of protease with xylanase and phytase (LOPRO) has potential in limiting FM use for tilapia grow-out feeds, however, the economic efficiencies were still below that of a 10% FM diet. Future considerations for research should target the indigestible dietary components in order to optimise enzyme dosages and maximise the benefits of each enzymes. In conclusion, a comparative life cycle assessment (LCA) was used to evaluate the environmental impacts of low FM diets and commercial feeds associated with tilapia production in Thailand. The study showed that the low FM enzyme supplemented diets had lower impact potentials and were environmental superior to the average (10% FM) commercial standard. LCA modules are recommended for least-cost formulation programmes as an option going forward. Additionally, LCA can be used as a predictive tool to guide farmers, especially small-scale producers, on the potential impacts of feed input choices and feeding practices. This will ensure higher product quality but also demonstrate environmental responsibility on the part of aquafeed and fish producers to final seafood consumers.

As endo-1,4-?-xilanases (EC 3.2.1.8) formam o maior grupo de enzimas hidrolíticas envolvido na degradação da xilana, visto que catalisam a hidrólise aleatória de ligações glicosídicas do tipo ?-1,4 no interior da sua cadeia principal, produzindo xilooligossacarídeos de diferentes tamanhos. Na natureza, essas enzimas estão intimamente relacionadas ao fornecimento de energia para o desenvolvimento dos organismos que as produzem. Em geral, as xilanases são isoladas preferencialmente de bactérias e fungos, e têm demonstrado grande potencial na produção de pães, ração animal, alimentos, bebidas, xilitol e bioetanol. O presente trabalho propôs o isolamento de uma nova endo-1,4-?-xilanase por meio de técnicas de produção e purificação acessíveis que pudessem viabilizar economicamente a integração desse biocatalisador aos processos industriais. O fungo Aspergillus tamarii Kita, oriundo de uma amostra de solo da Mata Atlântica, mostrou-se um bom produtor de xilanases em meio de cultura Adams suplementado com bagaço de cevada, um subproduto das indústrias cervejeiras. Após a otimização do processo de fermentação submersa, o extrato enzimático exibiu duas xilanases em gel de atividade para proteínas nativas, identificadas por espectrometria de massas como glicosil hidrolases pertencentes às famílias 10 e 11. A sacarificação enzimática de três resíduos agroindustriais, com base em um delineamento experimental de misturas, demonstrou que a combinação ternária desses componentes, em iguais proporções, possui considerável relevância para a produção de açúcares fermentáveis, tais como glicose e xilose. Em ensaios de imobilização, a xilanase GH11 foi satisfatoriamente estabilizada em matrizes de caráter iônico e covalente. A imobilização por ligação covalente multipontual em glioxil-agarose elevou a temperatura ótima de atividade de 60 para 65 °C e ofertou um considerável ganho de termoestabilidade ao derivado, que apresentou meia vida de 60 minutos a 80 °C. Além disso, a estabilização da enzima nesse suporte permitiu a produção dos seguintes xilooligossacarídeos: xilobiose, xilotriose, xilotetraose e xilopentaose. A purificação da xilanase GH11 foi realizada por meio de uma única etapa cromatográfica de troca catiônica, com rendimento final de 36,72% e um fator de purificação de 7,43 vezes. A massa molecular da enzima foi estimada em 19,5 kDa. Ademais, a sua estrutura tridimensional foi predita por modelagem comparativa, exibindo como modelo final uma arquitetura do tipo ?-jelly roll, comum às xilanases da família 11. Em ensaios de caracterização, a xilanase apresentou melhor atividade em pH 5,5 e manteve atividade residual superior a 80% na faixa de pH compreendida entre 4,0 e 9,0, durante 24 horas. Em relação à temperatura, a sua atividade ótima foi observada a 60 °C, contudo, a sua termoestabilidade foi mais expressiva a 50 °C, retendo cerca de 70% da sua atividade inicial por 480 minutos. Para a xilana beechwood, os valores de velocidade máxima e constante de dissociação aparente foram iguais a 1.330,20 µmol/min/mg e 8,13 mg/mL, respectivamente. Na concentração de 5 mM, os metais pesados Co2+, Hg+, Pb2+ e Zn2+ apresentaram um ponderável efeito de inibição sobre a xilanase GH11, enquanto que os íons Ba2+ e Ni2+, assim como os compostos ?-mercaptoetanol e DTT, exibiram um aumento superior a 20% em sua atividade. Por fim, a análise em tempo real da atividade xilanásica revelou que o substrato xilopentaose corresponde ao menor xilooligossacarídeo capaz de ser eficientemente hidrolisado. Sendo assim, a nova endo-xilanase GH11 isolada do fungo A. tamarii Kita exibe uma série de propriedades físico-químicas favoráveis a sua aplicabilidade em escala industrial. / The endo-1,4-?-xylanases (EC 3.2.1.8) form the largest group of hydrolytic enzymes involved in the degradation of xylan, since they catalyze the random hydrolysis of ?-1,4 glycosidic bonds within the main chain of this polysaccharide, producing xylooligosaccharides of different sizes. In nature, these enzymes are closely related to supplying energy for the development of the organisms that produce them. In general, xylanases are preferentially isolated from bacteria and fungi, which show great potential in industries as brewing, animal feed, food, beverage, xylitol and bioethanol. The present work proposed the isolation of a new endo-1,4-?-xylanase by available techniques of production and purification that can economically make feasible the integration of this biocatalyst to industrial processes. The fungus Aspergillus tamarii Kita, obtained from a soil sample of the Atlantic Forest, showed to be a good xylanase producer in Adams culture medium supplemented with barley bagasse, a byproduct of breweries. After the optimization of the submerged fermentation process, the crude enzymatic extract exhibited two xylanases in activity gel for native proteins, identified by mass spectrometry as glycosyl hydrolases belonging to families 10 and 11. The enzymatic saccharification of three agroindustrial residues, based on an experimental mixture design, showed that the ternary combination of these components, in equal proportions, has considerable relevance for the production of fermentable sugars, such as glucose and xylose. The xylanase GH11 was satisfactorily stabilized on matrices of ionic and covalent character in immobilization assays. Covalent multipoint immobilization on glyoxyl agarose raised its optimum temperature of activity from 60 to 65 °C and offered a considerable gain in thermostability to the derivative, which presented a half-life of 60 minutes at 80 °C. In addition, enzyme stabilization on this support allowed the production of the following xylooligosaccharides: xylobiose, xylotriose, xylotetraose and xylopentaose. Xylanase GH11 purification was carried out by means of a single cation exchange chromatographic step, with final yield of 36.72% and purification factor of 7.43 times. The molecular mass of this xylanase was estimated as 19.5 kDa. Moreover, its three-dimensional structure was predicted by comparative modeling, exhibiting a ?-jelly roll type folding as a final model, common to xylanases of family 11. In characterization tests, xylanase presented better activity at pH 5.5 and was considerably stable in the pH range of 4.0 to 9.0. Regarding temperature, its optimum activity was observed at 60 °C, however, its thermostability was more expressive at 50 °C, retaining about 70% of its initial activity for 480 minutes. In the presence of beechwood xylan, the values of maximum velocity and the constant of apparent dissociation were 1,330.20 µmol/min/mg and 8.13 mg/mL, respectively. At concentrations of 5 mM, the heavy metals Co2+, Hg+, Pb2+ and Zn2+showed an inhibition effect on the xylanase, whereas Ba2+ and Ni2+ ions, as well as ?-mercaptoethanol and DTT, exhibited an increase of more than 20% in their activity. Finally, the real-time analysis of xylanase activity revealed that the xylopentose substrate corresponds to the lowest xylooligosaccharide capable of being hydrolyzed. Thus, the new endo-xylanase GH11 isolated from the fungus A. tamarii Kita exhibits a series of physicochemical properties favorable to its applicability on an industrial scale.

O amido resistente de milho (ARM) não é digerido em humanos fornecendo benefícios para a saúde tais como redução do colesterol, do índice glicêmico e fermentação no cólon. Porém, a substituição parcial de farinha de trigo (FT) por ARM em massa de pão resulta na diluição do glúten prejudicando a qualidade do produto. Massa de pão foi produzida com 12,5 g/100g de ARM e os efeitos das enzimas glicose-oxidase (Gox), tranglutaminase (TG) e xilanase (HE) na massa foram estudados. Massa produzida sem ARM e sem enzimas foi considerada padrão e massa produzida com ARM e sem enzimas foi considerada controle para comparação. Uma metodologia foi desenvolvida para medir o torque durante o amassamento em grande escala, utilizando um reômetro dinâmico adaptado. As propriedades reológicas foram avaliadas nos testes de medidas descritivas de textura, adesividade Chen-Hoseney, extensão uniaxial Kieffer, extensão biaxial e testes oscilatórios em reômetro. Pão produzido de acordo com as formulações padrão, controle e ótima foi avaliado com relação ao volume específico (VEP), firmeza do miolo, cor e análise sensorial para o atributo preferência. As três enzimas testadas influenciaram positivamente o torque máximo atingido durante o amassamento que variou entre (8,36 e 9,38) N m. Gox e TG apresentaram efeito positivo na altura máxima desenvolvida pela massa medida em reofermentógrafo enquanto que o efeito da HE foi negativo. Uma formulação com ARM e enzimas apresentou desempenho de panificação similar a massa padrão (altura máxima ajustada igual a (45,5 ± 3,9) mm), correspondente a adição de (4, 2,5 e 0,5) mg/100g de TG, Gox e HE respectivamente (ótima). A formulação ótima apresentou adesividade, trabalho de adesão, coesividade, dureza, resiliência, resistência à extensão e extensibilidade similares a massa padrão e diferentes da massa controle. As enzimas aumentaram o índice de strain hardening reduzido pela adição de ARM. Para o pão de forma, o VEP variou entre (3,16 e 3,64) cm3/g (diferença não significativa) e o pão produzido com a formulação ótima foi o mais escolhido como preferido. Durante o armazenamento por até 7 dias, o ARM diminuiu a taxa de envelhecimento do pão enquanto que as enzimas apresentaram efeito oposto. Em geral, a substituição parcial de FT por ARM reduziu a elasticidade da massa diminuindo a qualidade do pão enquanto que as enzimas minimizaram esse efeito. / Resistant starch (RS) is not digested by humans providing benefits for health such as reduction of blood LDL cholesterol levels, reduction of post-prandial blood glucose and fermentation in the colon. However, partial replacement of wheat flour (WF) with RS prejudices bread quality due to gluten dilution. Dough was formulated with 12.5 g/100g of RS and the effects of the enzymes glucose-oxidase (Gox), tranglutaminase (TG) and xylanase (HE) were studied. Dough produced without RS or enzymes was considered as regular and dough produced with RS and without enzymes was considered as control for comparison. A methodology was developed to measure torque during dough mixing in large scale using a dynamic rheometer. With respect to dough rheology, tests performed included texture profile analysis, Chen-Hoseney stickiness, Kieffer uniaxial extension, biaxial extension and oscillatory tests in rheometer. Bread was produced from regular, control and optimum formulations and the quality was assessed concerning specific volume, firmness, color and preference by sensory panel. The three enzymes tested influenced positively the maximum torque during mixing which varied between (8.36 and 9.38) N m. Gox and TG showed positive effects on the maximum height developed by dough measured in rheofermentometer while HE showed a negative one. A formulation with RS and baking performance similar to regular dough was found (adjusted maximum height equal to (45.5 ± 3.9) mm), corresponding to (4, 2.5 and 0.5) mg/100g of TG, Gox and HE respectively (optimum). The optimum formulation showed stickiness, work of adhesion, cohesiveness, hardness, resilience, resistance to extension and extensibility, similar to the regular dough and statistically different from control dough. Strain hardening index was reduced by the addition of RS and increased by the addition of enzymes. Regarding bread tests, specific volume of bread varied between (3.16 e 3.64) cm3/g (not significant difference) and the bread produced with theoptimum dough was the most preferred by the sensory panel. During 7 days of storage, RS reduced the aging rate while enzymes had the opposite effect. In general, WF replacement by RS reduced dough elasticity affecting the bread quality while enzymes helped minimizing this effect.

Hemicelulose extraída do bagaço de cana-de-açúcar foi hidrolisada por enzimas de Thermoascus aurantiacus, Trichoderma reesei e Aspergilus niger para obtenção de xilo-oligossacarídeos (XOs). A hemicelulose foi extraída com hidróxido de sódio na presença de antraquinona, sulfito de sódio ou peróxido de hidrogênio. O uso de antraquinona ou sulfito aumentou o rendimento de extração, porém a hemicelulose apresentou baixa solubilidade em água, propriedade inadequada para a hidrólise enzimática. A extração da hemicellulose com peróxido de hidrogênio em meio alcalino foi otimizada através de um planejamento fatorial completo 24 variando-se a concentração de H2O2 de 2 a 6% (m/v), tempo de reação de 4 a 16 h, temperatura de 20 a 60°C e presença ou não de 0,5% de sulfato de magnésio. No ponto central o rendimento de extração de hemicelulose foi de 94,5% com remoção de mais que 88% da lignina. Um rendimento de 86% de hemicelulose com baixo teor de lignina (5,9%) foi obtido em 6% de peróxido de hidrogênio por 4h a 20°C. Nessa condição a hemicelulose apresentou massa molar de 21.000 g/mol, composição aproximada de 81% xilose, 4% de arabinose, 4% de glicose e 3% de ácidos urônicos, alta solubilidade em água (90 % em massa) e coloração amarelo claro. As enzimas usadas na hidrólise dessa hemicelulose foram produzidas pelo cultivo dos fungos em meio sólido contento farelo de trigo. Em todos os extratos foi observada baixa atividade de endoglucanase e &#946;-xilosidase e elevadas atividades de endo-&#946;-1,4-xilanase. A máxima atividade de xilanase foi produzida por T. aurantiacus (1500 U/g), enquanto A. niger produziu 500 U/g e T. reesei 240 U/g, em 5 dias de cultivo. O perfil de produção de XOs com enzimas de T. aurantiacus e T. reesei foi semelhante, o principal produto foi xilobiose, seguido por xilose, xilotriose, xilotetraose e xilopentaose, sendo esses XOs de cadeia linear. A hidrólise da hemicelulose com enzimas de A. niger produziu exclusivamente xilose, consequência da presença de elevada atividade de &#946;-xilosidase. A velocidade de conversão da hemicelulose em XOs com as enzimas de T. reesei foi maior no início da reação (6 h), diminuindo a partir de 24 h, período em que inicia a produção de xilose. A influência da concentração de substrato e carga de xilanase na conversão da hemicelulose em XOs foi avaliada através de um planejamento experimental 22 com face centrada. A condição otimizada da hidrólise (2,6% substrato e 60 U/g de endo-&#946;-1,4-xilanase) com o extrato de T. aurantiacus resultou em 42% de conversão em XOs. A otimização da hidrólise da hemicelulose com o extrato de T. reesei resultou em uma conversão máxima de 20%, com ótimo de 3,8 % de substrato e 87,5 U/g de endo-&#946;-1,4-xilanase. A eficiência da hidrólise com enzimas de T. aurantiacus foi maior que a obtida com alguns extratos comerciais testados neste trabalho. Além disso, apresentaram capacidade de degradar hemiceluloses de diferentes fontes: bétula e semente de aveia, com composições variadas. Diferenças na composição de açúcares e teor de lignina não interferiram na ação dessas enzimas. A hidrólise enzimática mostrou-se mais apropriada para a produção de XOs do que a auto-hidrólise, que gerou predominantemente xilose e houve formação de furfural. Apesar do curto tempo de reação, a produção de XOs foi menor e há necessidade de purificação para obtenção de um produto final com características desejáveis. / Hemicellulose extracted from sugarcane bagasse was hydrolyzed by enzymes from Thermoascus aurantiacus, Trichoderma reesei and Aspergilus niger to cause the degradation of xylan to xylooligosaccharides (XOs). Hemicellulose was extracted with hydrogen peroxide in the presence of antraquinone, sodium sulphite or hydrogen peroxide. Hemicelluloses extracted with antraquinone or sulphite presented low solubility in water, which is not appropriated to enzymatic hydrolysis. To maximize the hemicellulose yields several extraction conditions were examined applying the 24 factorial design: H2O2 concentration from 2 to 6% (w/v), reaction time from 4 to 16 h, temperature from 20 to 60°C, and magnesium sulfate absence or presence (0.5%, w/v). This approach allowed selection of conditions for the extraction of low and high lignin content hemicellulose. At midpoint the yield of hemicellulose was 94.5% with more than 88% of lignin removed. Hemicellulose in 86% yield with low lignin content (5.9%) was obtained with 6% H2O2 treatment for 4 h and 20°C. This hemicellulose is much lighter in color than samples obtained at the midpoint condition and was found suitable for subsequent enzymatic hydrolysis. The molecular weight of hemicellulose was 21,000 g/mol with composition of aproximately 81% xylose, 4% arabinose, 4% glucose and 3% uronic acids, high water solubility (90 %). Enzymes for hemicellulose hydrolysis were produced by the fungi on wheat bran. Cellulases and hemicellulases were present in all extracts especially the endo-&#946;-1,4-xylanase. The profile of production of XOs obtained on hydrolysis with enzymes from T. aurantiacus and T. reesei was similar, with the main product xylobiose, followed by xylose, xylotriose, xylotetraose and xylopentaose, and these XOs showed linear chain. The hydrolysis of hemicellulose with enzymes of A. niger produced exclusively xylose, a consequence of &#946;-xylosidase content. The rate of conversion of hemicellulose in XOs with enzymes of T. reesei was higher at the beginning of the reaction (6 h), decreasing from 24 h, when starts the production of xylose. The influence of substrate concentration and loading of xylanase in conversion of hemicellulose to XOs was evaluated by an 22 full factorial design with centered face. Optimization of hydrolysis (2.6% substrate and 60 U/g endo-&#946;-1,4-xylanase) with the extract of T. aurantiacus resulted in 42 % conversion XOs. The optimization with the extract of T. reesei resulted in a conversion of hemicellulose up to 20%, with optimal substrate 3.8% and 87.5 U/g endo-&#946;-1,4-xylanase. The efficiency of hydrolysis by enzymes from T. aurantiacus was superior to commercial extracts, and showed ability to degrade hemicelluloses of different compositions (birchwood and oat spelt). The structural differences, such as branches and lignin content did not affect the action of these enzymes. The differences in the efficiency and extent of enzymatic hydrolysis by enzymes of these fungi might have occurred in function of differences in physicochemical properties and specific activity. The enzymatic hydrolysis was more appropriate for production of XOs than autohydrolysis, which generated predominantly xylose and formation of furfural. Despite of short reaction time, the production of XOs was low and purification is needed in order to obtain a final product with desirable characteristics.

As endo-1,4-?-xilanases (EC 3.2.1.8) formam o maior grupo de enzimas hidrolíticas envolvido na degradação da xilana, visto que catalisam a hidrólise aleatória de ligações glicosídicas do tipo ?-1,4 no interior da sua cadeia principal, produzindo xilooligossacarídeos de diferentes tamanhos. Na natureza, essas enzimas estão intimamente relacionadas ao fornecimento de energia para o desenvolvimento dos organismos que as produzem. Em geral, as xilanases são isoladas preferencialmente de bactérias e fungos, e têm demonstrado grande potencial na produção de pães, ração animal, alimentos, bebidas, xilitol e bioetanol. O presente trabalho propôs o isolamento de uma nova endo-1,4-?-xilanase por meio de técnicas de produção e purificação acessíveis que pudessem viabilizar economicamente a integração desse biocatalisador aos processos industriais. O fungo Aspergillus tamarii Kita, oriundo de uma amostra de solo da Mata Atlântica, mostrou-se um bom produtor de xilanases em meio de cultura Adams suplementado com bagaço de cevada, um subproduto das indústrias cervejeiras. Após a otimização do processo de fermentação submersa, o extrato enzimático exibiu duas xilanases em gel de atividade para proteínas nativas, identificadas por espectrometria de massas como glicosil hidrolases pertencentes às famílias 10 e 11. A sacarificação enzimática de três resíduos agroindustriais, com base em um delineamento experimental de misturas, demonstrou que a combinação ternária desses componentes, em iguais proporções, possui considerável relevância para a produção de açúcares fermentáveis, tais como glicose e xilose. Em ensaios de imobilização, a xilanase GH11 foi satisfatoriamente estabilizada em matrizes de caráter iônico e covalente. A imobilização por ligação covalente multipontual em glioxil-agarose elevou a temperatura ótima de atividade de 60 para 65 °C e ofertou um considerável ganho de termoestabilidade ao derivado, que apresentou meia vida de 60 minutos a 80 °C. Além disso, a estabilização da enzima nesse suporte permitiu a produção dos seguintes xilooligossacarídeos: xilobiose, xilotriose, xilotetraose e xilopentaose. A purificação da xilanase GH11 foi realizada por meio de uma única etapa cromatográfica de troca catiônica, com rendimento final de 36,72% e um fator de purificação de 7,43 vezes. A massa molecular da enzima foi estimada em 19,5 kDa. Ademais, a sua estrutura tridimensional foi predita por modelagem comparativa, exibindo como modelo final uma arquitetura do tipo ?-jelly roll, comum às xilanases da família 11. Em ensaios de caracterização, a xilanase apresentou melhor atividade em pH 5,5 e manteve atividade residual superior a 80% na faixa de pH compreendida entre 4,0 e 9,0, durante 24 horas. Em relação à temperatura, a sua atividade ótima foi observada a 60 °C, contudo, a sua termoestabilidade foi mais expressiva a 50 °C, retendo cerca de 70% da sua atividade inicial por 480 minutos. Para a xilana beechwood, os valores de velocidade máxima e constante de dissociação aparente foram iguais a 1.330,20 µmol/min/mg e 8,13 mg/mL, respectivamente. Na concentração de 5 mM, os metais pesados Co2+, Hg+, Pb2+ e Zn2+ apresentaram um ponderável efeito de inibição sobre a xilanase GH11, enquanto que os íons Ba2+ e Ni2+, assim como os compostos ?-mercaptoetanol e DTT, exibiram um aumento superior a 20% em sua atividade. Por fim, a análise em tempo real da atividade xilanásica revelou que o substrato xilopentaose corresponde ao menor xilooligossacarídeo capaz de ser eficientemente hidrolisado. Sendo assim, a nova endo-xilanase GH11 isolada do fungo A. tamarii Kita exibe uma série de propriedades físico-químicas favoráveis a sua aplicabilidade em escala industrial. / The endo-1,4-?-xylanases (EC 3.2.1.8) form the largest group of hydrolytic enzymes involved in the degradation of xylan, since they catalyze the random hydrolysis of ?-1,4 glycosidic bonds within the main chain of this polysaccharide, producing xylooligosaccharides of different sizes. In nature, these enzymes are closely related to supplying energy for the development of the organisms that produce them. In general, xylanases are preferentially isolated from bacteria and fungi, which show great potential in industries as brewing, animal feed, food, beverage, xylitol and bioethanol. The present work proposed the isolation of a new endo-1,4-?-xylanase by available techniques of production and purification that can economically make feasible the integration of this biocatalyst to industrial processes. The fungus Aspergillus tamarii Kita, obtained from a soil sample of the Atlantic Forest, showed to be a good xylanase producer in Adams culture medium supplemented with barley bagasse, a byproduct of breweries. After the optimization of the submerged fermentation process, the crude enzymatic extract exhibited two xylanases in activity gel for native proteins, identified by mass spectrometry as glycosyl hydrolases belonging to families 10 and 11. The enzymatic saccharification of three agroindustrial residues, based on an experimental mixture design, showed that the ternary combination of these components, in equal proportions, has considerable relevance for the production of fermentable sugars, such as glucose and xylose. The xylanase GH11 was satisfactorily stabilized on matrices of ionic and covalent character in immobilization assays. Covalent multipoint immobilization on glyoxyl agarose raised its optimum temperature of activity from 60 to 65 °C and offered a considerable gain in thermostability to the derivative, which presented a half-life of 60 minutes at 80 °C. In addition, enzyme stabilization on this support allowed the production of the following xylooligosaccharides: xylobiose, xylotriose, xylotetraose and xylopentaose. Xylanase GH11 purification was carried out by means of a single cation exchange chromatographic step, with final yield of 36.72% and purification factor of 7.43 times. The molecular mass of this xylanase was estimated as 19.5 kDa. Moreover, its three-dimensional structure was predicted by comparative modeling, exhibiting a ?-jelly roll type folding as a final model, common to xylanases of family 11. In characterization tests, xylanase presented better activity at pH 5.5 and was considerably stable in the pH range of 4.0 to 9.0. Regarding temperature, its optimum activity was observed at 60 °C, however, its thermostability was more expressive at 50 °C, retaining about 70% of its initial activity for 480 minutes. In the presence of beechwood xylan, the values of maximum velocity and the constant of apparent dissociation were 1,330.20 µmol/min/mg and 8.13 mg/mL, respectively. At concentrations of 5 mM, the heavy metals Co2+, Hg+, Pb2+ and Zn2+showed an inhibition effect on the xylanase, whereas Ba2+ and Ni2+ ions, as well as ?-mercaptoethanol and DTT, exhibited an increase of more than 20% in their activity. Finally, the real-time analysis of xylanase activity revealed that the xylopentose substrate corresponds to the lowest xylooligosaccharide capable of being hydrolyzed. Thus, the new endo-xylanase GH11 isolated from the fungus A. tamarii Kita exhibits a series of physicochemical properties favorable to its applicability on an industrial scale.

Hemicelulose extraída do bagaço de cana-de-açúcar foi hidrolisada por enzimas de Thermoascus aurantiacus, Trichoderma reesei e Aspergilus niger para obtenção de xilo-oligossacarídeos (XOs). A hemicelulose foi extraída com hidróxido de sódio na presença de antraquinona, sulfito de sódio ou peróxido de hidrogênio. O uso de antraquinona ou sulfito aumentou o rendimento de extração, porém a hemicelulose apresentou baixa solubilidade em água, propriedade inadequada para a hidrólise enzimática. A extração da hemicellulose com peróxido de hidrogênio em meio alcalino foi otimizada através de um planejamento fatorial completo 24 variando-se a concentração de H2O2 de 2 a 6% (m/v), tempo de reação de 4 a 16 h, temperatura de 20 a 60°C e presença ou não de 0,5% de sulfato de magnésio. No ponto central o rendimento de extração de hemicelulose foi de 94,5% com remoção de mais que 88% da lignina. Um rendimento de 86% de hemicelulose com baixo teor de lignina (5,9%) foi obtido em 6% de peróxido de hidrogênio por 4h a 20°C. Nessa condição a hemicelulose apresentou massa molar de 21.000 g/mol, composição aproximada de 81% xilose, 4% de arabinose, 4% de glicose e 3% de ácidos urônicos, alta solubilidade em água (90 % em massa) e coloração amarelo claro. As enzimas usadas na hidrólise dessa hemicelulose foram produzidas pelo cultivo dos fungos em meio sólido contento farelo de trigo. Em todos os extratos foi observada baixa atividade de endoglucanase e &#946;-xilosidase e elevadas atividades de endo-&#946;-1,4-xilanase. A máxima atividade de xilanase foi produzida por T. aurantiacus (1500 U/g), enquanto A. niger produziu 500 U/g e T. reesei 240 U/g, em 5 dias de cultivo. O perfil de produção de XOs com enzimas de T. aurantiacus e T. reesei foi semelhante, o principal produto foi xilobiose, seguido por xilose, xilotriose, xilotetraose e xilopentaose, sendo esses XOs de cadeia linear. A hidrólise da hemicelulose com enzimas de A. niger produziu exclusivamente xilose, consequência da presença de elevada atividade de &#946;-xilosidase. A velocidade de conversão da hemicelulose em XOs com as enzimas de T. reesei foi maior no início da reação (6 h), diminuindo a partir de 24 h, período em que inicia a produção de xilose. A influência da concentração de substrato e carga de xilanase na conversão da hemicelulose em XOs foi avaliada através de um planejamento experimental 22 com face centrada. A condição otimizada da hidrólise (2,6% substrato e 60 U/g de endo-&#946;-1,4-xilanase) com o extrato de T. aurantiacus resultou em 42% de conversão em XOs. A otimização da hidrólise da hemicelulose com o extrato de T. reesei resultou em uma conversão máxima de 20%, com ótimo de 3,8 % de substrato e 87,5 U/g de endo-&#946;-1,4-xilanase. A eficiência da hidrólise com enzimas de T. aurantiacus foi maior que a obtida com alguns extratos comerciais testados neste trabalho. Além disso, apresentaram capacidade de degradar hemiceluloses de diferentes fontes: bétula e semente de aveia, com composições variadas. Diferenças na composição de açúcares e teor de lignina não interferiram na ação dessas enzimas. A hidrólise enzimática mostrou-se mais apropriada para a produção de XOs do que a auto-hidrólise, que gerou predominantemente xilose e houve formação de furfural. Apesar do curto tempo de reação, a produção de XOs foi menor e há necessidade de purificação para obtenção de um produto final com características desejáveis. / Hemicellulose extracted from sugarcane bagasse was hydrolyzed by enzymes from Thermoascus aurantiacus, Trichoderma reesei and Aspergilus niger to cause the degradation of xylan to xylooligosaccharides (XOs). Hemicellulose was extracted with hydrogen peroxide in the presence of antraquinone, sodium sulphite or hydrogen peroxide. Hemicelluloses extracted with antraquinone or sulphite presented low solubility in water, which is not appropriated to enzymatic hydrolysis. To maximize the hemicellulose yields several extraction conditions were examined applying the 24 factorial design: H2O2 concentration from 2 to 6% (w/v), reaction time from 4 to 16 h, temperature from 20 to 60°C, and magnesium sulfate absence or presence (0.5%, w/v). This approach allowed selection of conditions for the extraction of low and high lignin content hemicellulose. At midpoint the yield of hemicellulose was 94.5% with more than 88% of lignin removed. Hemicellulose in 86% yield with low lignin content (5.9%) was obtained with 6% H2O2 treatment for 4 h and 20°C. This hemicellulose is much lighter in color than samples obtained at the midpoint condition and was found suitable for subsequent enzymatic hydrolysis. The molecular weight of hemicellulose was 21,000 g/mol with composition of aproximately 81% xylose, 4% arabinose, 4% glucose and 3% uronic acids, high water solubility (90 %). Enzymes for hemicellulose hydrolysis were produced by the fungi on wheat bran. Cellulases and hemicellulases were present in all extracts especially the endo-&#946;-1,4-xylanase. The profile of production of XOs obtained on hydrolysis with enzymes from T. aurantiacus and T. reesei was similar, with the main product xylobiose, followed by xylose, xylotriose, xylotetraose and xylopentaose, and these XOs showed linear chain. The hydrolysis of hemicellulose with enzymes of A. niger produced exclusively xylose, a consequence of &#946;-xylosidase content. The rate of conversion of hemicellulose in XOs with enzymes of T. reesei was higher at the beginning of the reaction (6 h), decreasing from 24 h, when starts the production of xylose. The influence of substrate concentration and loading of xylanase in conversion of hemicellulose to XOs was evaluated by an 22 full factorial design with centered face. Optimization of hydrolysis (2.6% substrate and 60 U/g endo-&#946;-1,4-xylanase) with the extract of T. aurantiacus resulted in 42 % conversion XOs. The optimization with the extract of T. reesei resulted in a conversion of hemicellulose up to 20%, with optimal substrate 3.8% and 87.5 U/g endo-&#946;-1,4-xylanase. The efficiency of hydrolysis by enzymes from T. aurantiacus was superior to commercial extracts, and showed ability to degrade hemicelluloses of different compositions (birchwood and oat spelt). The structural differences, such as branches and lignin content did not affect the action of these enzymes. The differences in the efficiency and extent of enzymatic hydrolysis by enzymes of these fungi might have occurred in function of differences in physicochemical properties and specific activity. The enzymatic hydrolysis was more appropriate for production of XOs than autohydrolysis, which generated predominantly xylose and formation of furfural. Despite of short reaction time, the production of XOs was low and purification is needed in order to obtain a final product with desirable characteristics.

Made available in DSpace on 2017-05-12T14:47:16Z (GMT). No. of bitstreams: 1 Luciana_ Graciano.pdf: 1359694 bytes, checksum: 9321745e3e0faea5a21d91af3579b01a (MD5) Previous issue date: 2015-02-27 / The optimization of xylanase production of a new Aspergillus fumigatus Fresen strain (OI-1RT) was obtained by statistical approaches. The Plackett-Burman Design evaluation showed that the following components of Czapeck medium were biologically significant: sodium nitrate, potassium phosphate, magnesium sulfate and corn straw. These factors were selected to implement the 24 Central Composite Rotational Delineation with the proposed model validation. The response surface plots have indicated a trend for increased xylanase activity with increasing concentrations of maize straw. An additional test was carried out with different concentrations of maize straw and the optimized xylanase activity was 530 U ml-1 in the presence of 6.5% (w / v) of residual biomass, which was 11 times higher than the one obtained only with the Plackett-Burman Planning (45.8 U mL-1). The thermostability of the enzyme was kept at 90% at 50 °C for 6 hours. Enzyma tic hydrolyses tests were performed to obtain reducing sugars from maize straw, hydrolyzed maize straw and beechwood xylan. This procedure has been performed for 96 hours with 2 U ml-1 for xylanase (crude extract) and resulted in net production of 3.89, 20.96 and 21.64 &#956;mol mL-1 for reducing sugars, respectively. This indicated possible biotechnological applications to the crude extract with xylan-degrading enzymes (xylanase). / As xilanases são glicosídeo hidrolases (GHs) com diferentes propriedades físico-químicas e várias aplicações biotecnológicas, como na indústria têxtil, alimentícia e de papel e celulose. Xilanases podem ser utilizadas para a degradação de fontes de carbono renováveis, tais como os resíduos agroindustriais. Desta forma, é possível aproveitar a capacidade energética de compostos disponíveis em abundância e que poderiam ser descartados levando à poluição de solos e corpos hídricos. Atualmente, existe uma busca por estratégias que permitam a otimização de processos biotecnológicos para utilizar a capacidade energética presentes na biomassa para geração de produtos de valor agregado, como a produção de xilitol e do etanol celulósico. Ferramentas bioquímico-moleculares e planejamentos estatísticos de otimização de processos são exemplos de estratégias que visam melhorar o desempenho catalítico de enzimas. Neste contexto, este trabalho objetivou aproveitar resíduos agroindustriais para a indução de xilanases usando duas abordagens. Na primeira, foi realizada a clonagem e expressão do gene xynA1 (CCNA_02894) de Caulobacter crescentus em Escherichia coli com a obtenção de uma proteína recombinante fusionada a uma cauda de histidina carboxi-terminal (XynA1), que foi posteriormente purificada e caracterizada quanto a suas propriedades bioquímicas e cinéticas. Nesta análise, verificou-se que dentre as várias fontes de carbono testadas para indução da XynA1 em C. crescentus, os substratos palha de milho, palha de milho hidrolisada e o sabugo de milho foram os mais eficientes para induzir a atividade xilanásica. Além disso, a caracterização da XynA pura, obtida por cromatografia de afinidade em colunas préempacotadas de níquel-sepharose de elevado desempenho, mostrou que a XynA1 possui atividade enzimática e atividade específica de 18,26 U mL-1 e 2,22 e U mg-1usando xilano de beechwood como substrato na reação. A atividade de XynA1 foi inibida por EDTA e íons metálicos tais como Cu 2+ e Mg 2+. Em contraste, ß-mercaptoetanol, ditiotreitol (DTT), e Ca2+ induziram a atividade da enzima recombinante. Os dados cinéticos para XynA1 revelaram valores de KM e Vmáx de 3,77 mg mL-1 e 10,2 &#956;M min-1, respectivamente. Finalmente, a enzima apresentou um pH ótimo igual a 6 e temperatura ótima de 50 °C. Além disso, 80% da atividade de XynA1 foi mantida a 50 °C por 4 hor as de incubação. Isso sugere estabilidade térmica para aplicação em processos biotecnológicos. Na segunda etapa do trabalho, foi realizada a otimização da produção xilanásica por um novo isolado vii termotolerante de Aspergillus fumigatus Fresen. (OI-1R-T), obtido de bioma de Mata Atlântica, usando metodologias estatísticas de delineamento experimental como Planejamento Plackett-Burman (Plackett-Burman Design-PBD) e Delineamento Composto Central Rotacional (DCCR). Verificou-se, nesses ensaios, uma atividade xilanásica de 530 U mL-1 na presença de 6,5% de palha de milho no meio de cultivo otimizado. Ensaios de hidrólises enzimáticas da palha de milho, palha de milho hidrolisada e do xilano de beechwood foram realizados durante 96 horas com 2 U mL-1 de atividade xilanásica do extrato bruto otimizado no mesmo resíduo, resultando em uma produção líquida de 3,89, 20,96 e 21,64 &#956;mol mL-1 de açúcares redutores, respectivamente. Assim, sugere-se que a enzima fúngica, mesmo em extrato bruto, também poderia ser aplicada em processos biotecnológicos diversos.

Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2018 / Beer is one of the most widely consumed alcoholic beverages in the world. The brewing process is based on natural enzymatic activities that take place during the malting of barley grain, mashing of grist and fermentation of wort. Insufficient malt enzyme activity during the mashing process leads to high levels of barley β-glucan, arabinoxylan (AX) and dextrins in the wort as well as in the final beer. It was reported that high levels of β-glucan and AX increase wort and beer viscosity which lower the rate of beer filtration and this negatively affect the production rate in the brewery. During beer fermentation, brewing yeast catalyses the conversion of wort sugars to ethanol, carbon dioxide and other metabolic products. However, non-fermentable carbohydrates i.e., limit dextrins remain in the wort and final beer. These non-fermentable carbohydrates are known to contribute to the caloric value of beer which might lead to weight gain in consumers. The objectives of this study were to evaluate the effect of recombinant yeast strains expressing an endo-β-1,4-glucanase or an endo-β-1,4-xylanase on beer viscosity (as an indicator of filterability) and an α-amylase on residual sugars levels. The effect of the above mentioned enzymes on the aroma, appearance, flavour, mouth-feel and overall quality of the beer was also determined. Wort was produced in the University of Limpopo micro-brewery and the wort was pitched with different recombinant strains. The wild-type strain served as control. The results obtained showed that the xylanase expressing strain produced a measurable decrease in viscosity over the course of the fermentation, but endo-glucanase did not have any effect on the beer viscosity. The α-amylase producing strain, did not show a measurable reduction of residual sugars in the final beer probably as a result of very low activity on α-1,6 glycosidic bonds in dextrins during fermentation. The xylanase and α-amylase producing strain fermented effectively with good attenuation (decrease in wort specific gravity). The beer produced by the α-amylase and control strains were preferred in terms of taste and had similar qualities. The secreted amylolytic activity was not sufficient to significantly reduce residual sugar in the final beer. Although the xylanase secreting strain produced a beer with lower viscosity, the enzyme had a negative impact on the taste of the beer. Key words: Brewer’s yeast, beer fermentation, low calorie beer, amylase, xylanase, endo-glucanase.

Wood and wood products are of great economical and environmental importance, both in Sweden and globally. Biotechnology can be used both for achieving raw material of improved quality and for industrial processes such as biobleaching. Despite the enormous amount of carbon that is fixed as wood, the knowledge about the enzymes involved in the biosynthesis, re-organization and degradation of plant cell walls is relatively limited. In order to exploit enzymes more efficiently or to develop new biotechnological processes, it is crucial to gain a better understanding of the function and mechanism of the enzymes. This work has aimed to increase the knowledge about some of the enzymes putatively involved in the wood forming processes in Populus. Xyloglucan endotransglycosylases and a putative xylanase represent transglycosylating and hydrolytic enzymes, respectively. Carbohydrate binding modules represent non-catalytic modules, which bind to the substrate. Among 24 genes encoding for putative xyloglucan endotransglycosylases or xyloglucan endohydrolases that were identified in the Populus EST database, two were chosen for further studies (PttXTH16-34 and PttXTH16-35). The corresponding proteins, PttXET16-34 and PttXET16-35, were expressed in P. pastoris, purified and biochemically characterized. The importance of the N-glycans was investigated by comparing the recombinant wild-type proteins with their deglycosylated counterparts. In order to obtain the large amounts of PttXET16-34 that were needed for crystallization and development of biotechnological applications, the conditions for the large-scale production of PttXET16-34 in a fermenter were optimized. In microorganisms, endo-(1,4)-β-xylanases are important members of the xylan degrading machinery. These enzymes are also present in plants where they might fulfill a similar, but probably more restrictive function. One putative endo-(1,4)-β-xylanase, denoted PttXYN10A, was identified in the hybrid aspen EST library. Sequence analysis shows that this protein contains three putative carbohydrate-binding modules (CBM) from family 22 in addition to the catalytic module from GH10. Heterologous expression and reverse genetics were applied in order to elucidate the function of the catalytic module as well as the binding modules of PttXYN10A. Just as in microorganisms, some of the carbohydrate active enzymes from plants have one or more CBM attached to the catalytic module. So far, a very limited number of plant CBMs has been biochemically characterized. A detailed bio-informatic analysis of the CBM family 43 revealed interesting modularity patterns. In addition, one CBM43 (CBM43PttGH17_84) from a putative Populus b-(1,3)-glucanase was expressed in E. coli and shown to bind to laminarin (β-(1,3)-glucan), mixed-linked β-(1,3)(1,4)-glucans and crystalline cellulose. Due to their high specificity for different carbohydrates, CBMs can be used as probes for the analysis of plant materials. Generally, they are more specific than both staining techniques and carbohydrate-binding antibodies. We have used cellulose- and mannan binding modules from microorganisms as tools for the analysis of intact fibers as well as processed pulps. / QC 20100903

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 pPICZ&#945;-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.