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Orientador: Antonio Celso Pezzato / Resumo: O uso de enzimas que degradam polissacarídeos não-amiláceos (PNAs) em dietas para monogástricos tem sido cada vez mais explorado por nutricionistas, apesar dos mecanismos pelos quais estas enzimas melhoram o valor nutritivo dos alimentos ainda não estarem totalmente esclarecidos. Assim, este estudo foi desenvolvido com o objetivo de evidenciar os principais mecanismos de ação de enzimas que degradam PNAs e o efeito sobre frangos de corte alimentados com dietas à base de milho e farelo de soja. O primeiro experimento, relatado no Capítulo II, foi desenvolvido para avaliar os efeitos da inclusão de diferentes produtos enzimáticos compostos por xilanase e β-glucanase em dietas à base de milho com dois níveis de energia para frangos de corte sobre o desempenho, características intestinais, umidade de cama, incidência de lesões de pododermatite, e parâmetros bioquímicos do sangue. Foram utilizados 1.200 pintos machos, distribuídos em esquema fatorial 2 × 4 (dois níveis de energia × ausência ou inclusão de três diferentes xilanases + β-glucanases). O ganho de peso e a conversão alimentar foram prejudicados pelo fornecimento de dietas de baixa energia (CN; 2.925 kcal/kg na fase inicial e 3.025 kcal/kg na fase de crescimento). O uso de diferentes produtos enzimáticos compostos por xilanase e β-glucanase não afeta o desempenho e a qualidade intestinal de frangos de corte alimentados com dietas à base de milho. O experimento referente ao Capítulo III teve como objetivo avaliar o efeito... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

In recent years. as industrialized society developed. people have made a lot of environment pollution problems owing to overusing fossil fuel, moreover. fossil fuel is going to deplete. As the result. the study of the substitute energy is promoted. Ligonocellulose (lignin, cellulose and hemicelluloses are included) are the most plentiful renewable resources in the nature, and it have high economic values which extensively use on food, paper and energy. Recent years, it is a hot issue that decomposing cellulose into the minimum unit called glucose, and further, fermenting into alcohol to generate biomass energy. This research goal is cloning Arabidopsis endo-1,4-£]-glucanase and transfer to Escherichia coli(DH5£\). Selection pPICZ£\A the success transferr DNA Fragment. Then to sequence and because of cell in pPICZ£\A have expression in Pichia. pastoris. AOX1 promoter have Mass productions Arabidopsis endo-1,4-£]-glucanase gene in Pichia pastoris. Carries on the extracellular expression by the methyl alcohol induction way. Can obtain recombinant DNA protein. However the Western blotting analysis demonstration has this enzyme active protein At4g11050 pellet Molecular weight about 89 KDa. Again by way of congo red and Dye-CMC assay activeness of the examination enzyme protein. The result discovers At4g11050 in the pellet cell activity compares with negative control has the obvious activity.

Health benefits of high molecular weight (MW) β-glucans are well documented. Therefore, understanding and controlling depolymerization of β-glucan in baked products, would increase the effectiveness of β-glucan to confer health benefits. In this study we demonstrated that endogenous β-glucanase in wheat kernels are responsible for the depolymerization of β-glucans. A protocol was developed based on the Megzayme procedure to detect low levels of β-glucanase activity in wheat flour. This was confirmed by using HPLC-Calcofluor detection to monitor molecular weight changes. The distribution of β-glucanase in wheat kernels was also investigated. The effect of genotype, location, planting season and environmental factors on the level of endogenous β-glucanase in selected wheat cultivars was investigated using different wheat varieties planted under different condition and different seasons. Furthermore, kinetics of β-glucan depolymerization by endogenous glucanase in two dough systems with different moisture content was investigated. The results demonstrated that enzymes with β-glucanase activity are concentrated primarily in the outer layer of wheat kernels. Also genotype, environmental conditions and agronomic practice all had significant effects on the β-glucanase activity in wheat flours and poor harvesting conditions can significantly increase β-glucanase activity level in wheat. The kinetics results demonstrated that moisture content, incubation time and levels of endogenous β-glucanase activity of the system had significant impact on the final MW of β-glucan in the dough. Among all factors investigated, moisture content had the greatest impact. This study presents opportunities for industry to fortify baked products with high molecular weight β-glucan. / Ontario Ministry of Agricultural Food and Rural Affairs

Hydrolytic enzymes such as chitinases and glucanases are implicated in plant defense responses against fungal pathogens. These enzymes are responsible for the breakdown of chitin and glucan, two major components of the fungal cell walls. Genes encoding these enzymes have been used to genetically engineer plants to enhance their protection against fungal pathogens. Western Australia has over 4000 endemic plant species and a largely unknown fungal biota. Given that fungi possessing chitinases and glucanases with novel activities have been isolated in other parts of the world, we propose that fungi from Western Australian soils may possess novel biochemical/enzymatic activities. The aims of this research project were to isolate chitinolytic and glucanolytic fungi from soil and to clone the genes encoding for chitinase and glucanase enzymes. To achieve these aims, fungi with activity against chitin and glucan were isolated, the activity quantified by colorimetric and inhibition assays and gene fragments with homology to known chitinase and glucanase genes were isolated and their sequences determined. Soil fungi were isolated from five locations in and around the Perth Metropolitan area of Western Australia with the use of a medium containing Rose Bengal that eliminates all actinomycetes and most bacteria and reduces the growth of fast growing mold colonies. Forty-one isolates were obtained by this method. Twenty four chitinolytic and glucanolytic fungal isolates were identified by growing them on chitin-containing media to select for those species that utilised chitin/glucan as a carbon source. These were assayed for production of exo- and endochitinolytic and glucanolytic enzymes. Enzyme activity was compared between crude and dialysed supernatants. Exochitinase activity was determined in the supernatants of 4-day old fungal cultures by the release of p-nitrophenol from p-nitrophenyl-N-acetyl-â-D glucosaminide. The supernatants were measured for endochitinase activity determined by the reduction of turbidity of suspensions of colloidal chitin. Glucanase activity was determined by release of reducing sugar (glucose) from laminarin. Supernatants from eleven of the twenty four isolates showed significant levels of enzyme activity. Eleven isolates were assayed for activity against purified cell walls of phytopathogenic fungi. Activity was determined by measuring reducing sugars in the fungal supernatants against cell wall preparations of six economically important plant pathogens. Chitinolytic activity was detected in seven isolates against cell wall preparations of Botrytis cinerea and Rhizoctonia solani, in four isolates against Fusarium solani and Sclerotinia sclerotium; in five isolates against Ascochyta faba and in six isolates against Leptosphaeria maculans. Similarly glucanolytic activity was detected in eight isolates against B. cinerea, in seven against R. solani, in two against F. solani, in three against S. sclerotium and A. faba and in one against L. maculans. The supernatants derived from the isolates were used in a bioassay to determine growth inhibition against live B. cinerea spores by measuring turbidity reduction. Growth inhibition was measured against a control (B. cinerea, grown in medium with no added supernatant). Boiled supernatant did not inhibit the growth of B. cinerea spores but there was 100% inhibition by the crude supernatant from ten of the twenty four isolates. Similarly, supernatants were used to assess growth inhibition against live mycelia cultures of F. solani and S. sclerotium. Growth inhibition of F solani ranged from 9- 59%, boiled and crude supernatants respectively whilst growth inhibition of S. sclerotium ranged from 46-75%, boiled and crude supernatants respectively. Two partial chitinase genes from the soil filamentous ungus Trichoderma asperellum,(ChiA and ChiB) and a novel glucanase gene from the filamentous fungus Aspergillus (Glu1) were cloned. ChiA, was 639 bp long, encoding 191 amino acids with identity to other chitinase genes. Two highly conserved regions, characteristic of glycosyl hydrolases from family 18, were present. ChiB, was 887 bp long and encoded a 293 amino acid sequence that was closely related to an endochitinase gene from the filamentous fungus Trichoderma asperellum. The two highly conserved regions corresponding to the substrate binding and active sites that characterise the glycosyl hydrolases from family 18, also found in ChiA, were found in this gene. Glu1 was 2844 bp long and encoded a 948 amino acid sequence that shared high identity with a â-1, 3-glucanase from the filamentous fungus Aspergillus oryzae. The sequence contained conserved regions found in glycosyl hydrolases from family 17 that encode for substrate binding, N-terminal sequences and putative asparagine linked glycosylation sites. The partial putative sequence ChiA is probably a pseudogene because it has two inframe stop codons. However, once the entire sequence of ChiB is known, both ChiB and the novel glucanase gene Glu1 could be useful contenders for engineering resistance in crop plants.

Periplaneta americana apresenta em seu tubo digestivo pelo menos oito atividades β-glucanásicas distintas. Destas, cinco puderam ser purificadas até a homogeneidade e parcialmente caracterizadas. LAM (Mr=46.000) apresenta atividade exclusiva sobre laminarina, é inibida por altas concentrações de substrato e gera oligossacarídeos de baixo grau de polimerização (1 a 4) a partir de laminarina solúvel. LIQl (Mr=24.600) é capaz de hidrolisar laminarina e liquenana, produzindo oligossacarídeos de grau de polimerização 1, 2 e 4 a partir de laminarina solúvel e um único oligossacarídeo (grau de polimerização entre 3 e 4) a partir de liquenana. LIQ 2 (Mr= 22.300) é capaz de clivar laminarina e liquenana e é inibida por laminarina, clivando apenas ligações internas ao polímero. CELl e CEL2 (Mr=71.600 e 72.700) clivam liquenana e carboximetilcelulose, também atacando apenas ligações internas destes polissacarídeos. CELl e CEL2 também são capazes de atacar celulose cristalina. Todas as β-glucanases purificadas apresentam um pH ótimo em torno de 6,0, próximo ao pH luminal, e são relativamente estáveis em condições fisiológicas. Estas enzimas são purificadas a partir da fração solúvel do tubo digestivo do inseto em quantidades muito pequenas (até 7µg), o que inviabiliza sua caracterização estrutural refinada. Além destas proteínas, o sistema β-glucanásico de P. americana conta com duas celulases de baixo peso molecular (Mr= 15.000 e 17.000), e uma atividade ainda não caracterizada. Aparentemente, estas enzimas estão envolvidas na digestão incompleta da celulose e hemiceluloses ingeridas pelo inseto. LIQ 1 possui capacidade lítica sobre células de Saccharomyces cerevisiae, podendo estar envolvida na defesa do epitélio contra agentes infecciosos. / P. americana midgut has at least eight β-glucanases. Five were purified and partially characterized. LAM (Mr=46,000) is active only upon soluble laminarin, is inhibited by high amounts of substrate, and releases small oligosaccharides (1 to 4 glucoses). LIQ1 (Mr=24,600) is active upon laminarin and lichenan, releasing oligosaccharides with 1,2 and 4 glucosyl residues from soluble laminarin and only one oligossacharide, with a degree of polimerization between 3 and 4, from lichenan. LIQ2 (Mr=22,300) is active upon laminarin and lichenan and hydrolyzes only internal bond. CEL1 and CEL2 are active upon lichenan and carboxymethylcellulose, hydrolyzing internal bonds in these substrates. CEL1 and CEL2 also attack AVICEL. All β-glucanase activities have an optimum pH around 6.0 (near luminal pH) and are stable under physiological conditions. These enzymes are purified in very low amounts (up to 7µg from 10 animais). P. americana &#946,-glucanasic system also has two cellulases of low molecular weight (Mr= 15,000 and 17,000), and another not yet characterized. Probably these enzymes are involved in incomplete digestion of cellulose and hemicellulose ingested by the insect. LIQ 1 lyses Saccharomyces cerevisiae cells, and may be involved in epithelium defense against microorganisms.

Essa pesquisa avaliou o efeito da suplementação de dois complexos enzimáticos sobre o desempenho, incremento de energia e digestibilidade da matéria seca (MS), proteína (PB), aminoácidos (AAs) e extrato etéreo (EE) por 3 metodologias (coleta total de fezes, coleta com indicador e coleta ileal) em rações de frangos. No ensaio de desempenho foram utilizados 2.592 pintos machos distribuídos em um delineamento inteiramente casualizado com sete tratamentos (1: controle positivo; 2: controle negativo 1 (CN 1) decrescido de 2% de energia metabolizável aparente (EMA), AA e PB na fase de 1 a 21 dias e de 2,5% na fase de 22-42 dias; 3: controle negativo 2 (CN 2) decrescido de 4% de EMA, AA e PB na fase de 1 a 21 dias e de 5% na fase de 22-42 dias; 4: CN 1 mais 400 g do complexo A (α-amilase e β-glucanase); 5: CN 1 mais 500 g do complexo B (α-amilase, β-glucanase e xilanase); 6: CN 2 mais 400 g do complexo A; 7: CN 2 mais 500 g do complexo B), sendo o tratamento 1 com 6 repetições e os outros com 7, totalizando 48 parcelas experimentais com 54 aves cada. Foi adotado um programa alimentar com 2 fases: de 1 a 21 dias e de 22 a 42 dias. As características de desempenho avaliadas foram: consumo de ração, ganho de peso, conversão alimentar, conversão alimentar ajustada, viabilidade e índice de eficiência produtiva. No ensaio de metabolismo foram utilizados 351 pintos machos de um dia alojados em baterias seguindo delineamento inteiramente casualizado com os mesmos tratamentos do experimento 1, sendo os três primeiros com 5 repetições e os demais com 6, totalizando 39 parcelas experimentais com 9 aves por gaiola. O complexo enzimático B na dieta com menor redução proporcionou desempenho similar ao encontrado no controle positivo nas diferentes fases estudadas. Na primeira coleta do ensaio de metabolismo, realizada com aves de 21 dias, os complexos enzimáticos não foram eficazes em incrementar a energia da ração e a digestibilidade da matéria seca, do extrato etéreo, da proteína bruta e dos aminoácidos. Na coleta realizada com aves de 42 dias, o tratamento 5 proporcionou maiores valores de energia digestível e metabolizável, não diferindo dos tratamentos 1, 3 e 4 na metodologia de coleta total e dos tratamentos 3 e 4 nas metodologias de coleta com indicador e coleta ileal. O tratamento 5 ainda apresentou maior digestibilidade da MS pela metodologia de coleta com indicador. Os complexos enzimáticos melhoraram a digestibilidade do EE quando comparados aos seus respectivos tratamentos controle. Os tratamentos 4 e 5 apresentaram maior digestibilidade da PB pela metodologia de coleta ileal. Os maiores coeficientes de digestibilidade dos AAs concentraram-se no tratamento 5, seguido pelo tratamento 4. No geral, a metodologia de coleta total de excretas promoveu resultados mais consistentes. Conclui-se que a utilização de enzimas exógenas e viável técnica e economicamente. / This study evaluated the effect of supplementation of two enzymatic complexes on performance, energy increment and digestibility of dry matter (DM), crude protein (CP), amino acids (AA) and ether extract (EE) by three methodologies (total excreta collection, collection with marker and ileal collection) in broilers diets. In the performance assay a total of 2,592 male broiler chicks were randomly distributed in seven treatments (1: positive control; 2: negative control 1 (NC 1) with 2% reduction on apparent metabolizable energy (AME), AA and CP from 1 to 21 days and 2,5% from 22 to 42 days; 3: negatice control 2 (CN 2) with 4% reduction on AME, AA and CP from 1 to 21 days and 5% from 22 to 42 days; 4: NC 1 with 400 ppm of enzymatic complex A (α-amylase and β-glucanase); 5: NC 1 with 500 ppm of enzymatic complex B (α-amylase, β-glucanase and xylanase); 6: NC 2 with 400 ppm of enzymatic complex A; 7: NC 2 with 500 ppm of enzymatic complex B. Treatment one had six replicates and all the others treatments had seven replicates, in a total of 48 experimental units of 54 birds each. A feeding program with two phases was used from 1 to 21 days and from 22 to 42 days. Body weight gain, feed intake, feed conversion rate, adjusted feed conversion rate, viability and index of production efficiency were measured. In the metabolism assay a total of 351 male broiler chicks were housed in metallic batteries and randomly distributed in the same treatments of the performance assay. Treatments 1, 2 and 3 had 5 replicates and all the others treatments had 6 replicates, in a total of 39 experimental units of 9 birds by cage. The enzymatic complex B in the diet with lower reduction reached the same level of performance as the positive control diet. In the first collection of the metabolism assay (21 days), the enzymatic complexes were not efficient to increment the energy of the feed and digestibility of DM, EE, CP and AA. In the collection with 42 days, the treatment 5 provided the best result for digestible and metabolizable energies, but was not different of the treatments 1, 3 and 4 in total excreta collection methodology and of the treatments 3 and 4 in collection with marker and ileal collection methodologies. Treatment 5 even provided higher digestibility of DM by collection with marker methodology. The enzymatic complexes improved the ether extract digestibility when compared to their respective control treatments. The treatments 4 and 5 presented higher crude protein digestibility by ileal collection. The highest coefficients of digestibility of AA were observed in treatment 5, followed by treatment 4. In general, the total excreta collection methodology promoted higher consistency of the results. It was concluded that the exogenous enzymes utilization is technical and economically feasible.

β-1,3-glucanases são enzimas encontradas em muitos organismos, como fungos, bactéria e plantas. Suas funções incluem remodelamento de parede celular, defesa e digestão. É um alvo interessante para o controle populacional de insetos-praga, porque é ausente em vertebrados. Em insetos, é encontrada no intestino de muitas ordens diferentes, e hidrolizam β-1,3 ou β-1,3(4)-glucanas ingeridas, mas pouco se sabe sobre as propriedades e a função dessas enzimas. Nós estudamos três espécies de insetos, de três ordens diferentes. Spodoptera frugiperda (Lepidoptera), Tenebrio molitor (Coleoptera) e Abracris flavolineata (Orthoptera) são insetos herbívoros e pestes de plantações, mas suas beta-1,3-glucanases diferem significativamente. A β- 1,3-glucanase de S. frugiperda (SLAM) foi purificada do intestino médio da larva. Ela apresenta uma massa molecular de 37,5 kDa, um pH ótimo alcalino de 9,0, é ativa contra β-1,3-glucana (laminarina), mas é incapaz de hidrolizar β-1,3-1,6-glucana de levedura ou outros polisacarídeos. SLAM é não-processiva (0,4), e não é inibida por altas concentrações de substrato. Diferente de outras β-1,3-glucanases digestivas de insetos, SLAM é incapaz de lisar células de Saccharomyces cerevisae. O cDNA correspondente à SLAM foi clonado e sequenciado, demonstrando que a proteína pertence à família 16 das Glicosídeo-Hidrolases. A modelagem tridimensional de SLAM, feito com base em homologia de sequência, sugere que o resíduo E228 possa afetar a ionização dos resíduos catalíticos, causando o deslocamento do pH ótimo da enzima. Anti-corpos específicos para SLAM foram produzidos, e estes reagem com uma única proteína oriunda do intestino médio da larva, responsável pela atividade β-1,3- glucanásica majoritária. A imunocitolocalização de SLAM demonstra que a enzima é encontrada em vesículas secretórias e no glicocálix das células colunares, e portanto não é originária de simbiontes. Nós clonamos e sequenciamos o cDNA correspondente à β-1,3-glucanase majoritária presente no intestino médio da larva de T. molitor (TLAM). Ela pertence à família 16 das Glicosídeo-Hidrolases e está relacionada com proteínas ligantes de β -glucanas, da mesma forma que a enzima de S. frugiperda. A modelagem tridimensional por homologia de sequência permitiu identificar alguns resíduos de amino-ácidos (E174, E179, H204, Y304, R127 e R2181) no sítio ativo da enzima, que podem ser importantes para a atividade de TLAM. A β-1,3-glucanase digestiva do gafanhoto Abracris flavolineata (Orthoptera) é diferente das enzimas já estudadas em insetos. Ela apresenta uma estratégia catalítica processiva, liberando glicose como maior parte dos produtos, e é inibida por altas concentrações de substrato. Para estudar as bases estruturais desse mecanismo, nós procuramos obter a sequência de cDNA correspondente à enzima já caracterizada. O alinhamento múltiplo das β-1,3- glucanases de insetos e proteínas ligantes de beta-glucanas indicou que uma duplicação gênica da enzima do ancestral comum de moluscos e artrópodes. Uma cópia originou as beta-1,3-glucanase de insetos, perdendo uma região N-Terminal com cerca de 100 pares de bases, enquanto a outra cópia originou as proteínas ligantes de beta-glucana, perdendo os resíduos catalíticos. / β-1,3-glucanases are widespread enzymes, found in all major groups of invertebrates, fungi, bacteria and plants. Since this enzyme is absent in vertebrates, it constitutes an interesting target for control of insect pests population. Its functions range from cell wall remodeling, defense and digestion. In insects, it is found in the gut of many different orders, hydrolyzing β-1,3 or β-1,3(4)-glucanas, but little is known about the properties and function of these enzymes. We studied three insect species each pertaining to a different order. Spodoptera frugiperda (Lepidoptera), Tenebrio molitor (Coleoptera) and Abracris flavolineata are herbivores and crops pests, but their β-1,3- glucanases differ significantly. S. frugiperda β -1,3-glucanase (SLAM) was purified from the larval midgut. It has a molecular mass of 37.5 kDa, an alkaline optimum pH of 9.0, is active against β-1,3-glucan (laminarin), but cannot hydrolyze yeast β-1,3-1,6-glucan or other polysaccharides. The enzyme is an endoglucanase with low processivity (0.4), and is not inhibited by high concentrations of substrate. In contrast to other digestive β-1,3- glucanases from insects, SLAM is unable to lyse Saccharomyces cerevisae cells. The cDNA encoding SLAM was cloned and sequenced, showing that the protein belongs to glycosyl hydrolase family 16 as other insect glucanases and glucan-binding proteins. SLAM homology modeling suggests that E228 may affect the ionization of the catalytic residues, thus displacing the enzyme pH optimum. SLAM antiserum reacts with a single protein in the insect midgut. Immunocytolocalization reveals the presence of the enzyme in secretory vesicles and glycocalyx from columnar cells. We cloned and sequenced the cDNA of T. molitor β-1,3-glucanase. It belongs to glycoside hydrolase family 16, and is related to other insect glucanases and glucan-binding proteins. Sequence analysis and homology modeling allowed the identification of some residues (E174, E179, H204, Y304, R127 and R181) at the active site of the enzyme, which may be important for TLAM activity. The grasshopper A. flavolineata has a β-1,3-glucanase with a processive catalytic strategy. To study the structural basis of this property, we aimed to obtain its encoding sequence to better understand this catalytic mechanism. Multiple sequence alignment of insects β-1,3-glucanases and β-glucan-binding protein indicates that the β- 1,3-glucanase gene duplicated in the ancestor of mollusks and arthropods. One copy originated the insect β-1,3-glucanases after losing a 100 bp N-terminal portion and the arthropode β-glucan-binding proteins by the loss of the catalytic residues.

Essa pesquisa avaliou o efeito da suplementação de dois complexos enzimáticos sobre o desempenho, incremento de energia e digestibilidade da matéria seca (MS), proteína (PB), aminoácidos (AAs) e extrato etéreo (EE) por 3 metodologias (coleta total de fezes, coleta com indicador e coleta ileal) em rações de frangos. No ensaio de desempenho foram utilizados 2.592 pintos machos distribuídos em um delineamento inteiramente casualizado com sete tratamentos (1: controle positivo; 2: controle negativo 1 (CN 1) decrescido de 2% de energia metabolizável aparente (EMA), AA e PB na fase de 1 a 21 dias e de 2,5% na fase de 22-42 dias; 3: controle negativo 2 (CN 2) decrescido de 4% de EMA, AA e PB na fase de 1 a 21 dias e de 5% na fase de 22-42 dias; 4: CN 1 mais 400 g do complexo A (α-amilase e β-glucanase); 5: CN 1 mais 500 g do complexo B (α-amilase, β-glucanase e xilanase); 6: CN 2 mais 400 g do complexo A; 7: CN 2 mais 500 g do complexo B), sendo o tratamento 1 com 6 repetições e os outros com 7, totalizando 48 parcelas experimentais com 54 aves cada. Foi adotado um programa alimentar com 2 fases: de 1 a 21 dias e de 22 a 42 dias. As características de desempenho avaliadas foram: consumo de ração, ganho de peso, conversão alimentar, conversão alimentar ajustada, viabilidade e índice de eficiência produtiva. No ensaio de metabolismo foram utilizados 351 pintos machos de um dia alojados em baterias seguindo delineamento inteiramente casualizado com os mesmos tratamentos do experimento 1, sendo os três primeiros com 5 repetições e os demais com 6, totalizando 39 parcelas experimentais com 9 aves por gaiola. O complexo enzimático B na dieta com menor redução proporcionou desempenho similar ao encontrado no controle positivo nas diferentes fases estudadas. Na primeira coleta do ensaio de metabolismo, realizada com aves de 21 dias, os complexos enzimáticos não foram eficazes em incrementar a energia da ração e a digestibilidade da matéria seca, do extrato etéreo, da proteína bruta e dos aminoácidos. Na coleta realizada com aves de 42 dias, o tratamento 5 proporcionou maiores valores de energia digestível e metabolizável, não diferindo dos tratamentos 1, 3 e 4 na metodologia de coleta total e dos tratamentos 3 e 4 nas metodologias de coleta com indicador e coleta ileal. O tratamento 5 ainda apresentou maior digestibilidade da MS pela metodologia de coleta com indicador. Os complexos enzimáticos melhoraram a digestibilidade do EE quando comparados aos seus respectivos tratamentos controle. Os tratamentos 4 e 5 apresentaram maior digestibilidade da PB pela metodologia de coleta ileal. Os maiores coeficientes de digestibilidade dos AAs concentraram-se no tratamento 5, seguido pelo tratamento 4. No geral, a metodologia de coleta total de excretas promoveu resultados mais consistentes. Conclui-se que a utilização de enzimas exógenas e viável técnica e economicamente. / This study evaluated the effect of supplementation of two enzymatic complexes on performance, energy increment and digestibility of dry matter (DM), crude protein (CP), amino acids (AA) and ether extract (EE) by three methodologies (total excreta collection, collection with marker and ileal collection) in broilers diets. In the performance assay a total of 2,592 male broiler chicks were randomly distributed in seven treatments (1: positive control; 2: negative control 1 (NC 1) with 2% reduction on apparent metabolizable energy (AME), AA and CP from 1 to 21 days and 2,5% from 22 to 42 days; 3: negatice control 2 (CN 2) with 4% reduction on AME, AA and CP from 1 to 21 days and 5% from 22 to 42 days; 4: NC 1 with 400 ppm of enzymatic complex A (α-amylase and β-glucanase); 5: NC 1 with 500 ppm of enzymatic complex B (α-amylase, β-glucanase and xylanase); 6: NC 2 with 400 ppm of enzymatic complex A; 7: NC 2 with 500 ppm of enzymatic complex B. Treatment one had six replicates and all the others treatments had seven replicates, in a total of 48 experimental units of 54 birds each. A feeding program with two phases was used from 1 to 21 days and from 22 to 42 days. Body weight gain, feed intake, feed conversion rate, adjusted feed conversion rate, viability and index of production efficiency were measured. In the metabolism assay a total of 351 male broiler chicks were housed in metallic batteries and randomly distributed in the same treatments of the performance assay. Treatments 1, 2 and 3 had 5 replicates and all the others treatments had 6 replicates, in a total of 39 experimental units of 9 birds by cage. The enzymatic complex B in the diet with lower reduction reached the same level of performance as the positive control diet. In the first collection of the metabolism assay (21 days), the enzymatic complexes were not efficient to increment the energy of the feed and digestibility of DM, EE, CP and AA. In the collection with 42 days, the treatment 5 provided the best result for digestible and metabolizable energies, but was not different of the treatments 1, 3 and 4 in total excreta collection methodology and of the treatments 3 and 4 in collection with marker and ileal collection methodologies. Treatment 5 even provided higher digestibility of DM by collection with marker methodology. The enzymatic complexes improved the ether extract digestibility when compared to their respective control treatments. The treatments 4 and 5 presented higher crude protein digestibility by ileal collection. The highest coefficients of digestibility of AA were observed in treatment 5, followed by treatment 4. In general, the total excreta collection methodology promoted higher consistency of the results. It was concluded that the exogenous enzymes utilization is technical and economically feasible.

Made available in DSpace on 2014-07-29T15:16:28Z (GMT). No. of bitstreams: 1 Dissertacvao Silvio R C Barbosa 2008.pdf: 717228 bytes, checksum: d0896c3b0c9fa5a05f9d7107c2e59def (MD5) Previous issue date: 2008-05-30 / The fungus Sclerotinia sclerotiorum can interact with a great range of vegetable species as well as to obey to the discharge especificity and patogenic specialization . It is capable to digest the cellular wall of host plants using for such a series of biochemical mechanisms and morfogenetics that optimize the invasion. Several enzymes are produced during the interaction plant-host and among them they stand out the family of the poligalacturonases (PGs) and them beta glucanases. PGs catalyze the hydrolysis of the connection glycosídic bond - 1,4 and them beta glucanases liberate glucans and oligossacarídeos during the hydrolysis. Our work tried to characterize, besides the pH, the action of these enzymes, as well as the gene expression during the interaction with bean (Phaseolus vulgaris) and under different cultivation means, pectin 1%, wall extract 1% and glucose 1%. The activities were measured by the method DNS where the amount was measured of you sugar reducers in the middle and the gene expression through electrophoretic profiles analyzed after the technique of RT-PCR. The results showed variation of the activity of PGs in the interaction being the middle with pectin with larger expression of them. Them beta 1,3 and beta 1,4 glucanases were expressed in both culture means proposed, however there was larger production of beta 1,3 during the invasion. The variation of the expression of such enzymes in different culture means suggests complexity of specific biochemical roles for your production raising new approaches for the recognition of the roads that they promote the development of the disease / O fungo Sclerotinia sclerotiorum, pode interagir com uma grande gama de espécies vegetais bem como obedecer à alta especificidade e especialização patogênica. Ele é capaz de digerir a parede celular de plantas hospedeiras utilizando para tal uma série de mecanismos bioquímicos e morfogenéticos que otimizam a invasão. Várias enzimas são produzidas durante a interação planta-hospedeiro e dentre elas se destacam a família das poligalacturonases (PGs) e as beta glucanases. As PGs catalisam a hidrólise da ligação glicosídica α- 1-4 e as beta glucanases liberam glucanas e oligossacarídeos durante a hidrólise. Nosso trabalho procurou caracterizar, além do monitoramento do pH, a ação destas enzimas, bem como a expressão gênica durante a interação com feijão ( Phaseolus vulgaris ) e sob diferentes meios de cultivo, pectina a 1%, extrato de parede celular de plantas de feijoeiro a 1% e glicose 1%. As atividades foram medidas pelo método DNS onde mediu-se a quantidade de açucares redutores no meio e a expressão gênica através de perfis eletroforéticos analisados após a técnica de RT-PCR. Os resultados mostraram variação da atividade das PGs na interação sendo o meio com pectina com maior expressão delas. As beta 1,3 e beta 1,4 glucanases foram expressas em ambos os meios de cultura propostos, contudo houve maior produção de beta 1,3 durante a invasão. A variação da expressão de tais enzimas em diferentes meios de cultura sugerem complexidade de rotas bioquímicas específicas para sua produção suscitando novas abordagens para o reconhecimento dos caminhos que promovem o desenvolvimento da doença.