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- Efeito da suplementação enzimática, em uma dieta à base de cevada para frangos de carne, em diferentes períodos do seu crescimentoPublication . Cardoso, Vânia Alexandra da Silva; Ferreira, Luís Manuel dos Anjos; Lordelo, Maria Madalena dos SantosNeste trabalho, analisou-se a possibilidade de restringir a suplementação enzimática de uma dieta à base de cevada, a apenas alguns períodos de crescimento de frangos de carne. Foi utilizado um preparado multi-enzimático comercial (Rovabio®) com atividade sobre os componentes celulósicos e hemicelulósicos da parede celular. Os animais foram divididos por 4 tratamentos, ao 1º dia de idade. Os 4 tratamentos consistiram na alimentação das aves com dietas a) não suplementadas (CN) ou suplementadas com enzimas exógenas b) durante todo o período experimental (CP35), c) durante os primeiros 23 dias (CP23) ou d) os primeiros 11 dias do ensaio (CP11). Os dados revelaram que o desempenho produtivo, atividade enzimática gastrointestinal e tamanhos relativos dos órgãos abdominais, dos frangos alimentados com dietas suplementadas durante os 35 dias, foram idênticos (P<0.05) aos dos frangos alimentados com dietas suplementadas durante os 11 dias iniciais. Os desempenhos produtivos foram significativamente diferentes entre animais alimentados com dietas suplementadas e dietas não suplementadas. Adicionalmente, não foram encontradas diferenças significativas entre os três tratamentos suplementados (CP11, CP23 e CP35). Estes resultados sugerem que a ação de enzimas exógenas, usadas para suplementar dietas à base de cevada para frangos se restringe ao primeiro período do ciclo produtivo dos frangos.
- High-Throughput production and characterization of Carbohydrate-Active enZYmes for animal nutritionPublication . Lopes, Vânia Alexandra da Silva Cardoso; Fontes, Carlos Mendes Godinho de Andrade; Brás, Joana Luís ArmadaThe biodegradation of plant cell wall (PCW) carbohydrates is performed by microbial enzymes that are generally referred to as CAZymes. In animal nutrition, it is now well established that the monogastric animals produce a limited repertoire of CAZymes and as such cannot use efficiently some dietary ingredients that sometimes display antinutritional properties. The dietary supplementation with exogenous CAZymes improves the nutritive value of diets and increases animal’s performance. In particular, this study demonstrated that 1,3-1,4-β-glucanases and not cellulases improve the nutritive value of β-glucan-containing diets for monogastric animals. In addition, it was revealed that exogenous enzyme supplementation with β-xylanases improved the nutritive value of diets incorporating wheat lots with high viscosity and low endogenous endo-1,4-β-xylanase activity. In contrast, when the wheat lot showed lower viscosity and higher levels of endogenous endo-1,4-β-xylanase activity, broiler response was clearly diminished. Moreover, the data revealed that xylo-oligosaccharides released by xylanases acting on cereal arabinoxylans display a pre-biotic and positive effect in broiler chicks. However, although we observe an exponential accumulation of genomic and metagenomic information, knowledge on CAZYmes with potential to be used in animal nutrition is limited. This work also aimed to develop high-throughput (HTP) methodologies to isolate and characterize potentially important enzymes for animal nutrition. Thus, 1476 recombinant enzymes were selected and produced recombinantly. The data revealed that 79% of recombinant proteins were produced in the soluble form in Escherichia coli. Factors, such as, organism of origin, gene production strategy, fusion with solubility tags, protein molecular weight and amino acids composition of primary sequences may be used to justify and predict levels of solubility. The establishment of a high-throughput pipeline for recombinant enzyme production was used to obtain a library of feruloyl esterases (FAEs) and glucuronoyl esterases (GEs), enzymes which remove the side chains and break crosslinks between hemicellulosic carbohydrates and lignin. Thus 480 putative FAEs and 20 GEs were produced and biochemically characterized. Following gene isolation, 372 FAEs and 11 GEs were produced in a soluble form in E. coli. Activity results showed that 50% of the enzymes produced retained significant levels of activity and stability. The library of innovative FAEs and GEs produced during this project will be used to develop a novel generation of enzymes for animal nutrition, in particular to exploit the release of cellulose and hemicellulose from lignin.
- Recalcitrant cell wall of Ulva lactuca seaweed is degraded by a single ulvan lyase from family 25 of polysaccharide lyasesPublication . Costa, Monica; Pio, Luís Bernardo; Bule, Pedro; Duarte, Marlene; Alfaia, Cristina; Coelho, Diogo; Bras, Joana; Fontes, Carlos M.G.A.; Prates, José A.M; Cardoso, VâniaABSTRACT - Green macroalgae, e.g., Ulva lactuca, are valuable bioactive sources of nutrients; but algae recalcitrant cell walls, composed of a complex cross-linked matrix of polysaccharides, can compromise their utilization as feedstuffs for monogastric animals. This study aimed to evaluate the ability of pre-selected Carbohy- drate-Active enZymes (CAZymes) and sulfatases to degrade U. lactuca cell walls and release nutritive compounds. A databank of 199 recombinant CAZymes and sulfatases was tested in vitro for their action towards U. lactuca cell wall polysaccharides. The enzymes were incubated with the macroalga, either alone or in combination, to release reducing sugars and decrease fluorescence intensity of Calcofluor White stained cell walls. The individual action of a polysaccharide lyase family 25 (PL25), an ulvan lyase, was shown to be the most efficient in cell wall disruption. The ulvan lyase treatment, in triplicate measures, promoted the release of 4.54 g/L (P < 0.001) reducing sugars, a mono- and oligosaccharides release of 11.4 and 11.2 mmol/100 g of dried alga (P < 0.01), respectively, and a decrease of 41.7% (P < 0.001) in cell wall fluorescence, in comparison to control. The ability of ulvan lyase treatment to promote the release of nutritional compounds from alga biomass was also evaluated. A release of some monounsaturated fatty acids was observed, particularly the health beneficial 18:1c9 (P < 0.001). How- ever, no significant release of total fatty acids (P > 0.05), proteins (P ¼ 0.861) or pigments (P > 0.05) was found. These results highlight the capacity of a single recombinant ulvan lyase (PL25 family) to incompletely disrupt U. lactuca cell walls. This enzyme could enhance the bioaccessibility of U. lactuca bioactive products with promising utilization in the feed industry.
- Generation of a library of carbohydrate-active enzymes for plant biomass deconstructionPublication . Cardoso, Vânia; Bras, Joana L. A.; Costa, Ines F.; Ferreira, Luis M. A.; Gama, Luis; Vincentelli, Renaud; Henrissat, Bernard; Fontes, Carlos M.G.A.In nature, the deconstruction of plant carbohydrates is carried out by carbohydrate-active enzymes (CAZymes). A high-throughput (HTP) strategy was used to isolate and clone 1476 genes obtained from a diverse library of recombinant CAZymes covering a variety of sequence-based families, enzyme classes, and source organisms. All genes were successfully isolated by either PCR (61%) or gene synthesis (GS) (39%) and were subsequently cloned into Escherichia coli expression vectors. Most proteins (79%) were obtained at a good yield during recombinant expression. A significantly lower number (p < 0.01) of proteins from eukaryotic (57.7%) and archaeal (53.3%) origin were soluble compared to bacteria (79.7%). Genes obtained by GS gave a significantly lower number (p = 0.04) of soluble proteins while the green fluorescent protein tag improved protein solubility (p = 0.05). Finally, a relationship between the amino acid composition and protein solubility was observed. Thus, a lower percentage of non-polar and higher percentage of negatively charged amino acids in a protein may be a good predictor for higher protein solubility in E. coli. The HTP approach presented here is a powerful tool for producing recombinant CAZymes that can be used for future studies of plant cell wall degradation. Successful production and expression of soluble recombinant proteins at a high rate opens new possibilities for the high-throughput production of targets from limitless sources
- An individual alginate lyase is effective in the disruption of Laminaria digitata recalcitrant cell wallPublication . Costa, Monica; Pio, Luís Bernardo; Bule, Pedro; Cardoso, Vânia; Alfaia, Cristina; Coelho, Diogo; Brás, Joana; Fontes, Carlos M.G.A.; Prates, José A.MIn the present study, 199 pre-selected Carbohydrate-Active enZymes (CAZymes) and sulfatases were assessed, either alone or in combination, to evaluate their capacity to disrupt Laminaria digitata cell wall, with the consequent release of interesting nutritional compounds. A previously characterized individual alginate lyase, belonging to the family 7 of polysaccharide lyases (PL7) and produced by Saccharophagus degradans, was shown to be the most efcient in the in vitro degradation of L. digitata cell wall. The alginate lyase treatment, compared to the control, released up to 7.11 g/L of reducing sugars (p< 0.001) and 8.59 mmol/100 g dried alga of monosaccharides (p< 0.001), and reduced cell wall fuorescence intensity by 39.1% after staining with Calcofuor White (p= 0.001). The hydrolysis of gel-forming polymer alginate by the alginate lyase treatment could prevent the trapping of fatty acids and release benefcial monounsaturated fatty acids, particularly 18:1c9 (p < 0.001), to the extracellular medium. However, no liberation of proteins (p > 0.170) or pigments (p > 0.070) was observed. Overall, these results show the ability of an individual alginate lyase, from PL7 family, to partially degrade L. digitata cell wall under physiological conditions. Therefore, this CAZyme can potentially improve the bioavailability of L. digitata bioactive compounds for monogastric diets, with further application in feed industry.