RESUMO
Several biomaterials, including natural polymers, are used to increase cellular interactions as an effective way to treat bone injuries. Chitosan (CS) is one of the most studied biocompatible natural polymers. Graphene oxide (GO) is a carbon-based nanomaterial capable of imparting desired properties to the scaffolds. In the present study, CS and GO were used for scaffold preparation. CS was extracted from the mycelium of the fungus Aspergillus niger. On the other hand, GO was synthesized using an improved Hummers-Offemann method and was characterized by Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, atomic force microscopy (AFM), X-ray diffraction (XRD), and dynamic light scattering (DLS). Subsequently, three formulations (GO 0%, 0.5%, and 1%) were used to prepare the scaffolds by the freeze-drying technique. The scaffolds were characterized by FTIR, thermogravimetric analysis (TGA), and scanning electron microscopy (SEM), to determine their thermal stability and pore size, demonstrating that their stability increased with the increase of GO amount. Finally, the scaffolds were implanted, recollected 30 days later, and studied with an optical microscope, which evidenced the recovery of the tissue architecture and excellent biocompatibility. Hence, these results strongly suggested the inherent nature of chitosan/graphene oxide (CS/GO) scaffolds for their application in bone tissue regeneration.
Assuntos
Materiais Biocompatíveis/síntese química , Quitosana/química , Grafite/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Aspergillus niger/química , Materiais Biocompatíveis/química , Quitosana/isolamento & purificação , Liofilização , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Teste de Materiais , Microscopia Eletrônica de Varredura , Porosidade , Estabilidade Proteica , Ratos , Espectroscopia de Infravermelho com Transformada de Fourier , Termodinâmica , TermogravimetriaRESUMO
A fungal strain of Aspergillus niger was recovered from sediments collected in the Northeast coast of Brazil (Pecém's offshore port terminal). Cultivation in different growth media yielded a new ester furan derivative, 1, along with malformin A1, malformin C, cyclo (trans-4-hydroxy-L-Pro-L-Leu), cyclo (trans-4-hydroxy-L-Pro-L-Phe), cyclo (L-Pro-L-Leu), cyclo (L-Pro-L-Phe), pseurotin D, pseurotin A, chlovalicin, cyclo (L-Pro-L-Tyr) and cyclo (L-Pro-L-Val). Compound 1 was cytotoxic against HCT-116 cell line, showing IC50 = 2.9 µg/mL (CI 95% from 1.8 to 4.7 µg/mL).
Assuntos
Antineoplásicos/farmacologia , Aspergillus niger/química , Antineoplásicos/química , Brasil , Cicloexanonas/isolamento & purificação , Cicloexanonas/farmacologia , Dipeptídeos/isolamento & purificação , Dipeptídeos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Compostos de Epóxi/isolamento & purificação , Compostos de Epóxi/farmacologia , Furanos/química , Sedimentos Geológicos/microbiologia , Células HCT116 , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/isolamento & purificação , Peptídeos Cíclicos/farmacologia , Pirrolidinonas/isolamento & purificação , Pirrolidinonas/farmacologiaRESUMO
A new homology model of human microsomal epoxide hydrolase was derived based on multiple templates. The model obtained was fully evaluated, including MD simulations and ensemble-based docking, showing that the quality of the structure is better than that of only previously known model. Particularly, a catalytic triad was clearly identified, in agreement with the experimental information available. Analysis of intermediates in the enzymatic mechanism led to the identification of key residues for substrate binding, stereoselectivity, and intermediate stabilization during the reaction. In particular, we have confirmed the role of the oxyanion hole and the conserved motif (HGXP) in epoxide hydrolases, in excellent agreement with known experimental and computational data on similar systems. The model obtained is the first one that fully agrees with all the experimental observations on the system. Proteins 2017; 85:720-730. © 2016 Wiley Periodicals, Inc.
Assuntos
Inibidores Enzimáticos/química , Epóxido Hidrolases/química , Compostos de Epóxi/química , Microssomos Hepáticos/química , Simulação de Acoplamento Molecular , Ácido Valproico/análogos & derivados , Sequência de Aminoácidos , Aspergillus niger/química , Aspergillus niger/enzimologia , Domínio Catalítico , Sequência Conservada , Epóxido Hidrolases/antagonistas & inibidores , Epóxido Hidrolases/metabolismo , Compostos de Epóxi/metabolismo , Humanos , Cinética , Microssomos Hepáticos/enzimologia , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Alinhamento de Sequência , Streptomyces/química , Streptomyces/enzimologia , Homologia Estrutural de Proteína , Especificidade por Substrato , Ácido Valproico/químicaRESUMO
Recently, the crystal structure of the feruloyl esterase A from Aspergillus niger (AnFaeA) was elucidated. This enzyme displays an α/ß hydrolase fold and a catalytic triad similar to that found in fungal lipases (30-37% identity). Surprisingly, AnFaeA showed an overall fold similarity with the Rhizomucor miehei and other related fungal lipases. All these data strongly suggest that the ancestral function (lipase) had shifted, with molecular adaptation leading to a novel enzyme (type-A feruloyl esterase). The discovery of new feruloyl esterases could lead to get insight into the evolutionary pathways of these enzymes and into new possibilities of directed evolution of lipases. In this chapter, the production of Bacillus flexus NJY2 feruloyl esterases is described. Unlike the previously described feruloyl esterases, which mostly belong to eukaryotes (mainly fungus), this unique feruloyl esterases from a prokaryotic alkaliphile microorganism could be the starting point for new discoveries on lipase and feruloyl esterase evolutionary relationships.
Assuntos
Bacillus/química , Proteínas de Bactérias/química , Hidrolases de Éster Carboxílico/química , Motivos de Aminoácidos , Aspergillus niger/química , Proteínas de Bactérias/isolamento & purificação , Evolução Biológica , Hidrolases de Éster Carboxílico/isolamento & purificação , Domínio Catalítico , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Hidrólise , Cinética , Modelos Moleculares , Conformação Proteica , Rhizomucor/química , Especificidade por SubstratoRESUMO
Immobilization of lipases and phospholipases on, mainly, water insoluble carriers, helps in their economic reuse and in the development of continuous bioprocesses. Design of efficient lipases and phospholipases-immobilized system is rather a difficult task. A lot of research work has been done in order to optimize immobilization techniques and procedures and to develop an efficient immobilized system. A new rational design of immobilized derivatives strategy (RDID) has been conceived in favor of the successful synthesis of optimal lipases and phospholipases-immobilized derivatives, aiming prediction of the immobilized derivative's functionality and the optimization of load studies. RDID begins with the knowledge of structural and functional features of synthesis components (protein and carrier), and the practical goal of immobilized product. RDID was implemented in software named RDID ( 1.0 ). The employment of RDID allows selecting the most appropriate way to prepare immobilized derivatives more efficient in enzymatic bioconversion processes and racemic mixture resolution.
Assuntos
Enzimas Imobilizadas/química , Modelos Moleculares , Fosfolipases/química , Engenharia de Proteínas/métodos , Software , Algoritmos , Animais , Aspergillus niger/química , Aspergillus niger/enzimologia , Venenos de Abelha/química , Venenos de Abelha/enzimologia , Abelhas , Candida , Biologia Computacional , Venenos Elapídicos/química , Venenos Elapídicos/enzimologia , Elapidae , Lipase/química , Projetos de Pesquisa , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
Beta-glucan is a major component of fungal cell walls and shows various immunopharmacological activities including antitumor activity. Previously, we detected anti-beta-glucan antibody in human sera. Anti-beta-glucan antibody participates in the immune response to fungal cell wall beta-glucan. Patients on dialysis are at high risk of infection including fungal infections. We examined the plasma beta-glucan level and the titer of anti-beta-glucan antibody in dialysis patients. We measured plasma beta-1,3-glucan concentrations with the limulus G test and anti-beta-glucan antibody titers by ELISA with Candida beta-glucan-coated plates. We also examined the influence of the period of dialysis and the kind of dialysis membrane. The patients were positive for beta-1,3-glucan in their plasma. The anti-beta-glucan antibody titer was lower in the dialysis patients than in healthy volunteers. Long-term dialysis patients showed lower anti-beta-glucan antibody titers than short-term dialysis patients. No significant difference was found between the kinds of dialysis membrane. The titer of anti-beta-glucan antibody as recognition molecule of beta-glucan was low in dialysis patients compared with healthy volunteers. This is likely to be one factor explaining the sensitivity to infection of the dialysis patients. An appropriate application of culinary-medicinal mushroom such as Agaricus brasiliensis has potential for the prevention of fungal infection in dialysis patients.
Assuntos
Agaricus/imunologia , Anticorpos Antifúngicos/sangue , Parede Celular/imunologia , beta-Glucanas/sangue , beta-Glucanas/imunologia , Idoso , Anticorpos Antifúngicos/imunologia , Aspergillus niger/química , Aspergillus niger/imunologia , Candida/imunologia , Candida albicans/química , Candida albicans/imunologia , Feminino , Humanos , Falência Renal Crônica/imunologia , Falência Renal Crônica/microbiologia , Teste do Limulus , Masculino , Pessoa de Meia-Idade , Micoses/imunologia , Micoses/prevenção & controle , Diálise RenalRESUMO
Tannin acyl hydrolase, also known as tannase, is an enzyme with important applications in the food, feed, pharmaceutical, and chemical industries. However, despite a growing interest in the catalytic properties of tannase, its practical use is very limited owing to high production costs. Several studies have already demonstrated the advantages of solid-state fermentation (SSF) for the production of fungal tannase, yet the optimal conditions for enzyme production strongly depend on the microbial strain utilized. Therefore, the aim of this study was to improve the tannase production by a locally isolated A. niger strain in an SSF system. The SSF was carried out in packed-bed bioreactors using polyurethane foam as an inert support impregnated with defined culture media. The process parameters influencing the enzyme production were identified using a PlackettBurman design, where the substrate concentration, initial pH, and incubation temperature were determined as the most significant. These parameters were then further optimized using a Box-Behnken design. The maximum tannase production was obtained with a high tannic acid concentration (50 g/l), relatively low incubation temperature (30°C), and unique low initial pH (4.0). The statistical strategy aided in increasing the enzyme activity nearly 1.97-fold, from 4,030 to 7,955 U/l. Consequently, these findings can lead to the development of a fermentation system that is able to produce large amounts of tannase in economical, compact, and scalable reactors.
Assuntos
Aspergillus niger/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Proteínas Fúngicas/metabolismo , Microbiologia Industrial/métodos , Aspergillus niger/química , Aspergillus niger/metabolismo , Reatores Biológicos/microbiologia , Meios de Cultura/química , Meios de Cultura/metabolismo , Fermentação , Microbiologia Industrial/instrumentaçãoRESUMO
Colemanite is one of the most important underground riches of Turkey, having approximately 60 percent of the world boron deposits, and it has a large portion in the deposits. In this study, chemical leaching and biological leaching methods were used for production of boric acid from colemanite (2CaO · 3B3O3 · 5H2O) (Emet-Kütahya, Turkey). Oxalic acid concentration, temperature, stirring time and solid-to-liquid ratio were taken as parameters in the chemical leaching process. It was found that the dissolution rate increases with increasing oxalic acid concentration and temperature but it decreases at higher solid-to-liquid ratios in the chemical leaching process. Using optimum conditions (d100 = 0.075 mm; 5 percent solids by weight; 0.55 M oxalic acid; 80 +/- 2 ºC leaching temperature; 150 rpm stirring speed; 90 min leaching time) for colemanite sample (28.05 percent B2O3) on chemical leaching with oxalic acid experiments, the calculated boric acid extraction efficiency from colemanite ore was 97.89 percent. Optimum conditions on bioleaching of Emet-Kütahya, Turkey colemanite ores using the fungus Aspergillus niger were found to be as follows: reaction temperature 25 +/- 2ºC; solid-to-liquid ratio 5 percent solids by weight; d100 = 0.075 mm; stirring speed 150 rpm; initial the fungus populations in the inocula about 3 x 10(7) cells/ml and reaction time 21 days. The calculated boric acid extraction efficiency from colemanite ore was 90.18 percent under the optimum conditions. Bioleachate contained 12.95 g/l B2O3, 6.60 g/l Ca and 0.087 g/l Mg. Compared with chemical leaching at 5 percent pulp density, the fungus was less efficient in the extraction of B2O3 from colemanite but the difference in the extraction yields between the two processes was less than 10 percent. Although bioleaching generally requires a longer period of operation compared to chemical leaching, these results suggest that bioleaching by A...
Assuntos
Aspergillus niger/química , Boratos , Ácidos Bóricos , Cromatografia Líquida de Alta Pressão , Cinética , Minerais , Ácido Oxálico , Temperatura , Fatores de Tempo , TurquiaRESUMO
Existe un gran interés por el uso de enzimas lignocelulolíticas en varias industrias, y en la biodegradación de biomasa para la producción de biocombustibles y otras aplicaciones. Entre las fuentes microbianas de enzimas, Aspergillus niger es uno de los microorganismos más utilizados en la producción de enzimas industriales, debido a sus niveles altos de secreción de proteína y a su condición GRAS (generally regarded as safe). El objetivo del presente estudio fue evaluar la influencia de la concentración de inóculo en la morfología y producción de celulasas y xilanasas con A. niger en cultivo sumergido. Para ello, fueron inoculados matraces de 250 mL con 40 mL de medio con 3% (v/v) de una suspensión de 104 o 108 esporas por mililitro e incubados a 28 ºC y 175 rpm durante 120 horas. Se utilizaron 10 g*L-1 de lactosa como fuente de carbono. En cada caso se determinó la cantidad de biomasa, la proteína extracelular soluble, lactosa residual, actividad celulasa total y xilanasa cada 24 horas. Aunque no hubo un efecto notorio en la morfología de crecimiento, salvo en el color y el diámetro de pellets obtenidos, sí se afectó la µmax (0,06 y 0,03 h-1 para 104 y 108 esporas*mL-1, respectivamente) y la concentración máxima de biomasa. Además, mientras que las productividades volumétricas de celulasa (ΓFPA) (8,2 y 8,0 UI.*L-1*h-1 para 104 y 108 esporas*mL-1, respectivamente) fueron similares para ambos inóculos, la productividad de xilanasa (ΓXIL) fue mayor para el inóculo más concentrado (29,7 y 33,4 UI¨*L-1*h-1 para 104 y 108 esporas*mL-1, respectivamente). Los resultados indican que la productividad de celulasas y xilanasas está estrechamente relacionada con la concentración de inóculo.
There is a great interest for the use of lignocellulolytic enzymes in several industries and in biomass degradation for production of biofuels and other applications. Among the microbial sources of enzymes, Aspergillus niger is one of the most used microorganisms in the production of industrial enzymes due to its high levels of protein secretion and its GRAS (generally regarded as safe) condition. The aim of the present study was to evaluate the influence of A. niger inoculum concentration in the morphology and production of cellulases and xylanases in submerged cultures. For this, 250 mL flasks containing 40 mL culture medium were inoculated with a 3% (v/v) of either 104 or 108 spores per milliliter suspension and incubated at 28 º C and 175 rpm during 120 hours. Lactose (10 g*L-1) was used as the carbon source. In each case, the amount of biomass, the extracellular soluble protein, residual lactose, total celullase activity and xylanase activity were determined every 24 hours. Even thought there was not a notorious effect on the growth morphology, except in color and diameter of pellets; µmax was affected (0.06 and 0.03 h-1 for 104 and 108 spores*mL-1, respectively) as well as maximum biomass concentration. In addition, while the volumetric productivity of cellulase (8.2 and 8.0 UI*L-1*h-1 for 104 and 108 spores*mL-1, respectively) were similar for both inocula, the productivity of xylanase was greater for the more concentrated inoculum (29.7 and 33.4 UI*L-1*h-1 for 104 and 108 spores*mL-1, respectively).The results show that cellulase and xylanase productivities are closely related to the inoculum concentration.
Assuntos
Celulase/análise , Celulase/biossíntese , Celulase/genética , Celulase/imunologia , Celulase/química , Celulase/síntese química , Aspergillus niger/enzimologia , Aspergillus niger/fisiologia , Aspergillus niger/genética , Aspergillus niger/imunologia , Aspergillus niger/químicaRESUMO
True partitioning behaviour, which is independent of the protein concentration in aqueous two-phase systems, only occurs at relatively low protein concentration. The actual concentration limit depends on the properties of the protein. When the concentration of a protein exceeds relatively low values, precipitation at the interface can be observed. This protein precipitate is in equilibrium with the protein solubilized in each of the phases. This paper discusses the effect of protein solubility in view of the equilibrium of the protein concentration between the aqueous poly(ethylene glycol) and salt phases and the solid protein phase using three proteins. It was found that only rarely will the proteins be completely in solution as the concentration is increased until a solubility limit is reached and then the protein precipitates fully out of solution. A behaviour that came close to this was only seen in one case out of six. In virtually all cases, a third phase is formed which represents a solid aggregate phase which is in equilibrium with the other two, largely aqueous, phases. As the overall concentration of protein in the system is increased and the concentration in the top and bottom aqueous phases increases, the pseudo concentration in the solid-phase, C's, also increases. This could have interesting implications in terms of the amount of water associated with this phase and it certainly means that in this particular case, the solid phase is not a crystal.