RESUMO
Two strains of A. flavus one toxigenic (CECT 2687) and the other non-toxigenic (NRRL 6541) were studied for their genomic potential, growth capacity, and the production of enzymes on simple sugars, polysaccharides, and complex substrates under solid-state fermentation (SSF). According to the genome analysis, this fungus has many genes to degrade different types of polysaccharides and therefore it would be able to grow on different substrates. Both strains grow in all the carbon sources, but visibly CECT2687 grows slower than NRRL6541. However, we propose the growth index (GI) to establish a dry weight-diameter relationship as a more reliable measure that truly shows the growth preferences of the fungus. Considering this, the NRRL6541 shows less growth in 11 of the 16 evaluated carbon sources than CECT2687. Complex substrates were the best carbon source for the growth of both strains. Corncob (CC) induced the production of xylanases, pectinases, and almost all the accessory enzymes evaluated (except for α-xylosidase) this could make it an agricultural waste of interest to produce hemicellulolytic enzymes. Both strains produce a great variety of xylanases and pectinases (pathogenicity factors) making A. flavus a good potential candidate for the degradation of polysaccharides with a high content of xylan and pectin.
Assuntos
Aspergillus flavus , Endo-1,4-beta-Xilanases/biossíntese , Proteínas Fúngicas/biossíntese , Pectinas/metabolismo , Poligalacturonase/biossíntese , Xilanos/metabolismo , Aspergillus flavus/enzimologia , Aspergillus flavus/crescimento & desenvolvimento , Carbono/metabolismo , Especificidade da EspécieRESUMO
Aspergillus flavus was isolated from soil and exhibited laccase activity under both constitutive and copper induced conditions. Spiking the medium with 1 mM copper sulfate resulted in an increase in the activity which reached 51.84 U/mL, a distinctive protein band was detected at 60 kDa. The extracellular enzyme was purified 81 fold using gel filtration chromatography and resulted in two different laccase fractions L1 and L2, the latter had a higher enzymatic activity which reached 79.57 U/mL and specific activity of 64.17 U/µg protein. The analysis of the spectrum of the L2 fraction showed a shoulder at 330 nm which is characteristic for T2/T3 copper centers; both copper and zinc were detected suggesting that this is an unconventional white laccase. Primers of laccase gene were designed and synthesized to recover specific gene from A. flavus . Sequence analysis indicated putative laccase (Genbank ID: JF683612) at the amino acid level suggesting a close identity to laccases from other genera containing the copper binding site. Decolorization of textile waste water under different conditions showed possible application in bioremediation within a short period of time. The effect of copper on A. flavus was concentration dependent.
Assuntos
Aspergillus flavus/efeitos dos fármacos , Aspergillus flavus/enzimologia , Cobre/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Lacase/biossíntese , Ativação Transcricional/efeitos dos fármacos , Aspergillus flavus/genética , Aspergillus flavus/isolamento & purificação , Cromatografia em Gel , Meios de Cultura/química , DNA Fúngico/genética , Eletroforese em Gel de Poliacrilamida , Resíduos Industriais , Lacase/química , Lacase/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Microbiologia do Solo , Análise Espectral , Purificação da ÁguaRESUMO
Aspergillus flavus was isolated from soil and exhibited laccase activity under both constitutive and copper induced conditions. Spiking the medium with 1 mM copper sulfate resulted in an increase in the activity which reached 51.84 U/mL, a distinctive protein band was detected at 60 kDa. The extracellular enzyme was purified 81 fold using gel filtration chromatography and resulted in two different laccase fractions L1 and L2, the latter had a higher enzymatic activity which reached 79.57 U/mL and specific activity of 64.17 U/μg protein. The analysis of the spectrum of the L2 fraction showed a shoulder at 330 nm which is characteristic for T2/T3 copper centers; both copper and zinc were detected suggesting that this is an unconventional white laccase. Primers of laccase gene were designed and synthesized to recover specific gene from A. flavus. Sequence analysis indicated putative laccase (Genbank ID: JF683612) at the amino acid level suggesting a close identity to laccases from other genera containing the copper binding site. Decolorization of textile waste water under different conditions showed possible application in bioremediation within a short period of time. The effect of copper on A. flavus was concentration dependent.
Assuntos
Aspergillus flavus/efeitos dos fármacos , Aspergillus flavus/enzimologia , Cobre/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Lacase/biossíntese , Ativação Transcricional/efeitos dos fármacos , Aspergillus flavus/genética , Aspergillus flavus/isolamento & purificação , Cromatografia em Gel , Meios de Cultura/química , DNA Fúngico/genética , Eletroforese em Gel de Poliacrilamida , Resíduos Industriais , Lacase/química , Lacase/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Microbiologia do Solo , Análise Espectral , Purificação da ÁguaRESUMO
Aspergillus flavus was isolated from soil and exhibited laccase activity under both constitutive and copper induced conditions. Spiking the medium with 1 mM copper sulfate resulted in an increase in the activity which reached 51.84 U/mL, a distinctive protein band was detected at 60 kDa. The extracellular enzyme was purified 81 fold using gel filtration chromatography and resulted in two different laccase fractions L1 and L2, the latter had a higher enzymatic activity which reached 79.57 U/mL and specific activity of 64.17 U/μg protein. The analysis of the spectrum of the L2 fraction showed a shoulder at 330 nm which is characteristic for T2/T3 copper centers; both copper and zinc were detected suggesting that this is an unconventional white laccase. Primers of laccase gene were designed and synthesized to recover specific gene from A. flavus. Sequence analysis indicated putative laccase (Genbank ID: JF683612) at the amino acid level suggesting a close identity to laccases from other genera containing the copper binding site. Decolorization of textile waste water under different conditions showed possible application in bioremediation within a short period of time. The effect of copper on A. flavus was concentration dependent.(AU)
Assuntos
Aspergillus flavus , Aspergillus flavus/enzimologia , Cobre/metabolismo , Regulação Enzimológica da Expressão Gênica , Aspergillus flavus/genética , Aspergillus flavus/isolamento & purificação , Cromatografia em Gel , Meios de Cultura/químicaRESUMO
Sphingomyelinases D (SMases D) or dermonecrotic toxins are well characterized in Loxosceles spider venoms and have been described in some strains of pathogenic microorganisms, such as Corynebacterium sp. After spider bites, the SMase D molecules cause skin necrosis and occasional severe systemic manifestations, such as acute renal failure. In this paper, we identified new SMase D amino acid sequences from various organisms belonging to 24 distinct genera, of which, 19 are new. These SMases D share a conserved active site and a C-terminal motif. We suggest that the C-terminal tail is responsible for stabilizing the entire internal structure of the SMase D Tim barrel and that it can be considered an SMase D hallmark in combination with the amino acid residues from the active site. Most of these enzyme sequences were discovered from fungi and the SMase D activity was experimentally confirmed in the fungus Aspergillus flavus. Because most of these novel SMases D are from organisms that are endowed with pathogenic properties similar to those evoked by these enzymes alone, they might be associated with their pathogenic mechanisms.
Assuntos
Corynebacterium pseudotuberculosis/enzimologia , Fungos/enzimologia , Ixodes/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Aranhas/enzimologia , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Aspergillus flavus/enzimologia , Aspergillus flavus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Corynebacterium pseudotuberculosis/classificação , Corynebacterium pseudotuberculosis/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/classificação , Fungos/genética , Ixodes/classificação , Ixodes/genética , Modelos Moleculares , Dados de Sequência Molecular , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Filogenia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Esfingomielinas/química , Esfingomielinas/metabolismo , Aranhas/classificação , Aranhas/genéticaRESUMO
Agaricus brasiliensis CS1, Pleurotus ostreatus H1 and Aspergillus flavus produced holocellulases when grown in solid and submerged liquid cultures containing agro-industrial residues, including sugar cane bagasse and dirty cotton residue, as substrates. These isolates proved to be efficient producers of holocellulases under the conditions used in this screening. Bromatological analysis of agro-industrial residues showed differences in protein, fiber, hemicellulose, cellulose and lignin content. Maximal holocellulase activity (hemicellulase, cellulase and pectinase) was obtained using solid-state cultivation with 10% substrate concentration. In this case, remarkably high levels of xylanase and polygalacturonase activity (4,008 and 4,548 IU/l, respectively) were produced by A. flavus when grown in media containing corn residue, followed by P. ostreatus H1 with IU/l values of 1,900 and 3,965 when cultivated on 5% and 10% sugar cane bagasse, respectively. A. brasiliensis CS1 showed the highest reducing sugar yield (11.640 mg/ml) when grown on medium containing sugar cane bagasse. A. brasiliensis was also the most efficient producer of protein, except when cultivated on dirty cotton residue, which induced maximal production in A. flavus. Comparison of enzymatic hydrolysis of sugar cane bagasse and dirty cotton residue by crude extracts of A. brasiliensis CS1, P. ostreatus H1 and A. flavus showed that the best reducing sugar yield was achieved using sugar cane bagasse as a substrate.
Assuntos
Agricultura , Celulase/biossíntese , Fungos/enzimologia , Fungos/crescimento & desenvolvimento , Resíduos Industriais , Plantas/microbiologia , Agaricus/enzimologia , Agaricus/crescimento & desenvolvimento , Aspergillus flavus/enzimologia , Aspergillus flavus/crescimento & desenvolvimento , Carboidratos/análise , Celulase/metabolismo , Misturas Complexas , Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/análise , Hidrólise , Oxirredução , Pleurotus/enzimologia , Pleurotus/crescimento & desenvolvimentoRESUMO
Growth and enzymes production by Aspergillus flavipes FP-500 were evaluated on pectin, polygalacturonic acid, galacturonic acid, arabinose, rhamnose, xylose, glycerol and glucose at different initial pH values. We found that the strain produced exopectinases, endopectinases and pectin lyases. Exopectinases and pectin lyase were found to be produced at basal levels as constitutive enzymes and their production was modulated by the available carbon source and pH of culture medium and stimulated by the presence of inducer in the culture medium. Endo-pectinase was basically inducible and was only produced when pectin was used as carbon source. Our results suggest that pectinases in A. flavipes FP-500 are produced in a concerted way. The first enzyme to be produced was exopectinase followed by Pectin Lyase and Endo-pectinase.
Avaliou-se o crescimento e a produção de enzimas por Aspergillus flavipes FP-500 em pectina, ácido poligalacturônico, ácido galacturônico, arabinose, ramnose, xilose, glicerol e glicose, em diferentes valores de pH inicial. Verificamos que a cepa produziu exopectinases, endopectinases e pectina liases. Exopectinases e pectina liases foram produzidas em níveis basais como enzimas constitutivas e sua produção foi modulada pela fonte de carbono disponível e pelo pH do meio de cultura e estimulada pela presença de indutores no meio de cultura. Endopectinase foi indutível e produzida somente quando pectina foi utilizada como fonte de carbono. Nossos resultados sugerem que as pectinases de A. flavipes FP-500 são produzidas de forma planejada. A primeira enzima a ser produzida foi expopectinase, seguida por pectina liase e endopectinase.
Assuntos
Aspergillus flavus/crescimento & desenvolvimento , Aspergillus flavus/enzimologia , Pectinas/análise , Poligalacturonase/análise , Métodos , MétodosRESUMO
Investigations were carried out to optimize the culture conditions for the production of xylanase and beta-xylosidase by Aspergillus flavus, a filamentous fungus isolated from soil. The production of enzymes was tolerant to a wide range of initial culture pH values. Maximum xylanase (190 U/ml) and beta-xylosidase (35 U/ml) production was obtained when the strain was grown on mineral medium supplemented with 3% (w/v) corn cob powder as the carbon source. The enzymes had optimal activities at pH values between 5.5 and 6.0 and exhibited high activity and stability under alkaline conditions.