Tracking the connection between evolutionary and functional shifts using the fungal lipase/feruloyl esterase A family

BACKGROUND: There have been many claims of adaptive molecular evolution, but what role does positive selection play in functional divergence? The aim of this study was to test the relationship between evolutionary and functional shifts with special emphasis on the role of the environment. For this purpose, we studied the fungal lipase/feruloyl esterase A family, whose functional diversification makes it a very promising candidate. RESULTS: The results suggested functional shift following a duplication event where neofunctionalisation of feruloyl esterase A had occurred with conservation of the ancestral lipase function. Evolutionary shift was detected using the branch-site model for testing positive selection on individual codons along specific lineages. Positively selected amino acids were detected. Furthermore, biological data obtained from site-directed mutagenesis experiments clearly demonstrated that certain amino acids under positive selection were involved in the functional shift. We reassessed evolutionary history in terms of environmental response, and hypothesized that environmental changes such as colonisation by terrestrial plants might have driven adaptation by functional diversification in Euascomycetes (Aspergilli), thus conferring a selective advantage on this group. CONCLUSION: The results reported here illustrate a rare example of connection between fundamental events in molecular evolution. We demonstrated an unequivocal connection between evolutionary and functional shifts, which led us to conclude that these events were probably linked to environmental change

Homologous expression of the feruloyl esterase B gene from Aspergillus niger and characterization of the recombinant enzyme

The faeB gene encoding the feruloyl esterase B (FAEB) was isolated from Aspergillus niger BRFM131 genomic DNA. The faeB gene, with additional sequence coding for a C-terminal histidine tag, was inserted into an expression vector under the control of the gpd promoter and trpC terminator and expressed in a protease deficient A. niger strain. Homologous overproduction allows to reach an esterase activity of 18 nkat mL(-1) against MCA as substrate. The improvement factor was 16-fold higher as compared to the production level obtained with non-transformed A. niger strain induced by sugar beet pulp. The corresponding secretion yield was estimated to be around 100 mg L(-1). Recombinant FAEB was purified 14.6-fold to homogeneity from an 8-day-old culture by a single affinity chromatographic step with a recovery of 64%. SDS-PAGE revealed a single band with a molecular mass of 75 kDa, while under non-denatured conditions, native enzyme has a molecular mass of around 150 kDa confirming that the recombinant FAEB is a homodimer. The recombinant and native FAEB have the same characteristics concerning temperature and pH optima, i.e., 50 degrees C and 6, respectively. In addition, the recombinant FAEB was determined to be quite stable up to 50 degrees C for 120 min. Kinetic constants for MCA, MpCA, and chlorogenic acid (5-O-caffeoyl quinic acid) were as follows: Km: 0.13, 0.029, and 0.16 mM and Vmax: 1101, 527.6, and 28.3 nkat mg(-1), respectively. This is the first report on the homologous overproduction of feruloyl esterase B in A. niger

Controladoria : desafios e vantagens para o pequeno e médio empreendedor

Orientador: Vicente PachecoMonografia(Especialização) - Universidade Federal do Paraná,Setor de Ciências Sociais Aplicadas, Curso de Especialização em ControladoriaResumo: A constante busca pelo sucesso empresarial promove a revisão dos métodos de gestão utilizados pelas empresas e incentiva a busca por novas técnicas e ferramentas que auxiliem as organizações na tomada de decisão. Ao procurar informações de como melhor administrar um negócio, o gestor se depara com uma quantidade considerável de livros, artigos e outros meios de comunicação que fornecem conhecimento sobre os métodos e formas de obtenção de lucro e garantir assim a continuidade da empresa. Estes podem ser os maiores objetivos de uma empresa, mas se não houver planejamento e monitoramento a tendência e não alcançar sua meta principal. Assim, a procura por um único método que englobe todas as questões necessárias dentro de uma organização, em termos operacionais e estratégicos, tem aumentado a cada dia. A Controladoria surge assim como um grande diferencial para as empresas que visam o aprimoramento continuo e profissionais devidamente capacitados para exercer a função controller estão sendo cada vez mais procurados. Diante da realidade brasileira e perfil prevalecente das empresas no país, e importante verificar se as organizações possuem condições de permanecerem no mercado competitivo e sem a necessidade de elevados investimentos, principalmente em informação. Uma analise sobre as vantagens e desvantagens quando da implementação da controladoria em uma pequena e media empresa e necessária devido a forte relação entre as variáveis custo e beneficia. Assim, este trabalho foi elaborado no intuito de verificar se a controladoria como ferramenta de suporte a gestão pode ser adaptada a qualquer tipo de empresa e analisar se sua utilização e viável para o pequeno e media empreendedor

Chromogenic substrates for feruloyl esterases

International audienceChromogenic mono- and diferuloyl-butanetriol analogs were prepared by chemical syntheses and their efficiency was evaluated as substrates for feruloyl esterases from Aspergillus nige

Exploration of members of Aspergillus sections nigri, flavi, and terrei for feruloyl esterase production

International audienceAbstract: The ability of members of Aspergillus sections Nigri, Flavi, and Terrei to produce feruloyl esterases was studied according to their substrate specificity against synthetic methyl esters of hydroxycinnamic acids. Type A feruloyl esterases (FAEA), induced during growth on cereal-derived products, show a preference for the phenolic moiety of substrates that contain methoxy substitutions, as found in methyl sinapinate, whereas type B feruloyl esterases (FAEB) show a preference for the phenolic moiety of substrates that contain hydroxyl substitutions, as occurs in methyl caffeate. All the strains of Aspergillus section Nigri (e.g., A. niger and A. foetidus) were able to produce feruloyl esterases with activity profiles similar to those reported for FAEA and FAEB of A. niger when grown on oat–spelt xylan and sugar beet pulp, respectively. The two genes encoding these proteins, faeA and faeB, were identified by Southern blot analysis. The strains of Aspergillus sections Flavi (e.g., A. flavus, A. flavo-furcatus, and A. tamarii) and Terrei (e.g., A. terreus) were able to produce type A and type B enzymes. faeA was revealed in genomic DNA of these strains, and FAEA was determined by immunodetection in cultures grown in oat–spelt xylan. In addition, type B enzymes, not related to faeB, were efficiently induced by oat–spelt xylan and exhibited very original activity profiles on sugar beet pulp. This work confirms that the members of the genus Aspergillus are good feruloyl esterase producers.La capacité des espèces du genre Aspergillus sections Nigri, Flavi et Terrei à produire des féruloyl estérases a été étudiée en fonction de leur spécificité vis-à-vis d'hydroxycinnamates de méthyle synthétiques. Les féruloyl estérases de type A (FAEA), induites lors de croissance sur des produits céréaliers, sont connues pour montrer une préférence pour les moitiés aromatiques des substrats comportant des groupements méthoxy, comme le sinapinate de méthyle, tandis que les féruloyl estérases de type B (FAEB), induites lors de croissance sur pulpes de betterave, montrent une préférence pour les moitiés aromatiques des substrats comportant plusieurs groupements hydroxyle, comme l'acide caféique. Toutes les souches d'Aspergillus section Nigri (i.e., A. niger et A. foetidus) ont produit des féruloyl estérases avec des profils d'activité similaires à ceux de la FAEA et de la FAEB, dans des cultures réalisées en présence de xylane d'avoine ou de pulpe de betterave, respectivement. Les deux gènes codant ces protéines, faeA et faeB, ont été détectés par Southern blot. Par ailleurs, les souches d'Aspergillus appartenant aux sections Flavi (i.e., A. flavus, A. flavo-furcatus et A. tamarii) et Terrei (i.e., A. terreus) se sont avérées capables de produire des enzymes de types A et B. Le gène faeA a été retrouvé dans l'ADN génomique de ces souches et la protéine FAEA a été détectée dans les cultures réalisées en présence de xylane d'avoine. De plus, les enzymes de type B ont été fortement induites par le xylane d'avoine et ont montré des profils d'activités très originaux dans les cultures contenant des pulpes de betterave comme inducteur. Ces travaux confirment que le genre Aspergillus est d'un grand intérêt pour la production de féruloyl estérases

Chromogenic substrates for feruloyl esterases

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Purification and characterization of a chlorogenic acid hydrolase from Aspergillus niger catalysing the hydrolysis of chlorogenic acid

International audienceAmong 15 Aspergillus strains, Aspergillus niger BRFM 131 was selected for its high chlorogenic acid hydrolase activity. The enzyme was purified and characterized with respect to its physico-chemical and kinetic properties. Four chromatographic steps were necessary to purify the protein to homogeneity with a recovery of 2%. Km of the chlorogenic acid hydrolase was estimated to be 10 μM against chlorogenic acid as substrate. Under native conditions, the protein presented a molecular mass of 170 kDa, and SDS-PAGE analysis suggested the presence of two identical 80 kDa subunits. Isoelectric point was 6.0; pH optimum for activity was determined to be 6.0 and temperature optima to be 55 °C. The N-terminal sequence did not present any homology with other cinnamoyl ester hydrolases previously described suggesting the purification of a new protein. The chlorogenic acid hydrolase was used successfully for the production of caffeic acid, which possesses strong antioxidant properties, from natural substrates specially rich in chlorogenic acid like apple marc and coffee pulp

Respective importance of protein folding and glycosylation in the thermal stability of recombinant feruloyl esterase A

International audienceThe thermal stability of four molecular forms (native, refolded, glycosylated, non-glycosylated) of feruloyl esterase A (FAEA) was studied. From the most to the least thermo-resistant, the four molecular species ranked as follows: (i) glycosylated form produced native, (ii) non-glycosylated form produced native, (iii) non-glycosylated form produced as inclusion bodies and refolded, and (iv) glycosylated form produced native chemically denatured and then refolded. On the basis of these results and of crystal structure data, we discuss the respective importance of protein folding and glycosylation in the thermal stability of recombinant FAEA

Feruloyl esterases as a tool for the release of phenolic compounds from agro-industrial by-products

Agro-industrial by-products are a potential source of added-value phenolic acids with promising applications in the food and pharmaceutical industries. Here two purified feruloyl esterases from Aspergillus niger, FAEA and FAEB were tested for their ability to release phenolic acids such as caffeic acid, p-coumaric acid and ferulic acid from coffee pulp, apple marc and wheat straw. Their hydrolysis activity was evaluated and compared with their action on maize bran and sugar beet pulp. The specificity of both enzymes against natural and synthetic substrates was evaluated; particular attention was paid to quinic esters and lignin monomers. The efficiency of both enzymes on model substrates was studied. We show the ability of these enzymes to hydrolyze quinic esters and ester linkages between phenolic acids and lignin monomer

Quantitative study of lipase secretion, extracellular lipolysis, and lipid storage in the yeast Yarrowia lipolytica grown in the presence of olive oil: analogies with lipolysis in humans

International audienceLipase secretion, extracellular lipolysis, and fatty acid uptake were quantified in the yeast Yarrowia lipolytica grown in the presence of olive oil and/or glucose. Specific lipase assays, Western blot analysis, and ELISA indicated that most of the lipase activity measured in Y. lipolytica cultures resulted from the YLLIP2 lipase. Lipase production was triggered by olive oil and, during the first hours of culture, most of the lipase activity and YLLIP2 immunodetection remained associated with the yeast cells. YLLIP2 was then released in the culture medium before it was totally degraded by proteases. Olive oil triglycerides were largely degraded when the lipase was still attached to the cell wall. The fate of lipolysis products in the culture medium and inside the yeast cell, as well as lipid storage, was investigated simultaneously by quantitative TLC-FID and GC analysis. The intracellular levels of free fatty acids (FFA) and triglycerides increased transiently and were dependent on the carbon sources. A maximum fat storage of 37.8% w/w of yeast dry mass was observed with olive oil alone. A transient accumulation of saturated FFA was observed whereas intracellular triglycerides became enriched in unsaturated fatty acids. So far, yeasts have been mainly used for studying the intracellular synthesis, storage, and mobilization of neutral lipids. The present study shows that yeasts are also interesting models for studying extracellular lipolysis and fat uptake by the cell. The quantitative data obtained here allow for the first time to establish interesting analogies with gastrointestinal and vascular lipolysis in humans