RESUMO
OBJECTIVES: The aim of the present work was to perform the co-culture between Trichoderma longibrachiatum LMBC 172, a mesophilic fungus, with Thermothelomyces thermophilus LMBC 162, a thermophilic fungus, by submerged fermentation in a bioreactor. RESULTS: There was an increase in protein production, reaching the value of 35.60 ± 3.76 µg/ml at 72 h. An increase in the amount of proteins of 27.5% in relation to the isolated cultivation of T. longibrachiatum and 19.7% in comparison when T. thermophilus was isolated and cultivated. After that, the saccharification profile of three varieties of sugarcane (sugarcane in natura, culms of sugarcane SP80-3280, and culms of Energy cane) submitted in two pretreatments (autohydrolysis and chemical) was performed. The (e) chemical pretreatment was the better in generating of fermentable sugars from sugarcane bagasse and culms of Energy cane, while with the autohydrolysis pretreatment was obtained the better values to culms of SP80-3280 sugarcane. The sugars found were glucose, xylose, arabinose, and cellobiose. CONCLUSION: These results suggest that the co-culture between these microorganisms has the potential to produce an enzymatic cocktail with high performance in the hydrolysis of materials from the sugar-alcohol industry.
Assuntos
Hypocreales , Saccharum , Celulose/química , Técnicas de Cocultura , Hypocreales/metabolismo , Glucose/metabolismo , Fermentação , HidróliseRESUMO
Climate change is predicted to cause more extreme events, such as heatwaves, and different precipitation patterns. The effects of warming and short-term drought on soil microbial communities, in particular fungal communities, remain largely unexplored under field conditions. Here, we evaluated how the fungal community of a tropical grassland soil responds to these changes. A field experiment was carried out in a temperature free-air controlled enhancement (T-FACE) facility in Ribeirão Preto, Brazil. The isolated and combined effects of drought and a 2°C increase in temperature were investigated. Based on metabarcoding of the ITS2 region, a total of 771 operational taxonomic units were observed. While warming affected the community structure, drought affected the alpha diversity, and the interaction between warming and drought affected both diversity and structure. The change in community composition driven by warming affected only the less abundant species (>1% of the total sequences). The aspect of the fungal communities that was most affected was diversity, which was increased by drought (p < .05), mostly by reducing the dominance of a single species, as observed in the watered plots. In a phylogenetic context, some fungal taxa were favoured by changes in temperature (Hypocreales) and drought (Sordariales) or disadvantaged by both (Pleosporales). It was of note that a water deficit increased the abundance of phytopathogenic fungi, such as Curvularia, Thielavia and Fusarium species. Overall, our results provide evidence that fungal communities in tropical grassland soils have greater sensitivity to drought than to temperature, which might increase the incidence of certain soil-borne diseases.
Assuntos
Micobioma , Solo , Brasil , Mudança Climática , Secas , Pradaria , Micobioma/genética , Filogenia , Microbiologia do SoloRESUMO
This paper describes a bioprocess to obtain omegas-6 and 9 from the hydrolysis of Açaí (Euterpe oleracea Martius) and Buriti (Mauritia flexuosa) oils by lipases immobilized on octyl-sepharose. For this, oils and butters were initially selected as the carbon source which resulted in higher production of lipases in Beauveria bassiana and Fusarium oxysporum cultures. The carbon source that provided secretion of lipase by B. bassiana was Açaí oil, and for F. oxysporum, Bacuri butter. Lipases obtained under these conditions were immobilized on octyl-sepharose, and both, the derivatives and the crude extracts were biochemically characterized. It was observed that the immobilization promoted an increase of stability in B. bassiana and F. oxysporum lipase activities at the given temperatures and pH. In addition, the immobilization promoted hyperactivation of B. bassiana and F. oxysporum lipase activities being 23.5 and 11.0 higher than free enzyme, respectively. The hydrolysis of Açaí and Buriti oils by the derivatives was done in a biphasic (organic/aqueous) system, and the products were quantified in RP-HPLC. The results showed the potential of these immobilized lipases to obtain omegas-6 and 9 from Brazilian natural oils. This work may improve the enzymatic methodologies for obtaining foods and drugs enriched with fatty acids.
Assuntos
Arecaceae/química , Carotenoides/química , Euterpe/química , Lipase/química , Óleos de Plantas/química , Carbono/química , Cromatografia Líquida , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas em TandemRESUMO
Phytases catalyze the cleavage of phosphate groups from phytic acid. Here, we have studied the effects of glycosylation on the properties of Aspergillus japonicus C03 phytase expressed in Pichia pastoris. The enzyme ORF of 1338 nucleotides was cloned from genomic DNA, and encoded a secreted mature protein of 446 amino acids, which included the sequence motif RHGXRX and dipeptide HD, classifying the phytase as a histidine acid phosphate. After transformation and 72h of induction, P.pastoris GS115 expressed a 75kDa protein showing 526U/mg phytase activity and 143mg/L of protein. The amino acid sequence showed 8 and 3 potential N- and O-glycosylation sites, respectively. Analysis by ESMS showed two glycoform masses of 75,467 and 72,793, which after deglycosylation decreased to 54,327 and 54,128, respectively, indicating a carbohydrate content of 27-30%. A single GlcNAc was assigned at Asn6, Asn38, Asn84, Asn99, Asn209, Asn218, Asn355 and Asn367. The recombinant phytase showed maximum activity at 50°C, a half-life of 40min, and farUVCD spectroscopy indicated a secondary structure rich in α-helix. Thermal denaturation analyses reveal the melting temperature varied from 50°C at pH 6 to a maximum of 66°C at pH 3 and pH 4.
Assuntos
6-Fitase/metabolismo , Clonagem Molecular/métodos , Fosfatase Ácida , Sequência de Aminoácidos , Aspergillus/genética , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Modelos Moleculares , Pichia/genética , Desnaturação Proteica , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
Endophytic fungi, mostly belonging to the Ascomycota, are found in the intercellular spaces of the aerial plant parts, particularly in leaf sheaths, sometimes even within the bark and root system without inducing any visual symptoms of their presence. These fungi appear to have a capacity to produce a wide range of enzymes and secondary metabolites exhibiting a variety of biological activities. However, they have been only barely exploited as sources of enzymes of industrial interest. This review emphasizes the suitability and possible advantages of including the endophytic fungi in the screening of new enzyme producing organisms as well as in studies aiming to optimize the production of enzymes through well-known culture processes. Apparently endophytic fungi possess the two types of extracellular enzymatic systems necessary to degrade the vegetal biomass: (1) the hydrolytic system responsible for polysaccharide degradation consisting mainly in xylanases and cellulases; and (2) the unique oxidative ligninolytic system, which degrades lignin and opens phenyl rings, comprises mainly laccases, ligninases and peroxidases. The obvious ability of endophytic fungi to degrade the complex structure of lignocellulose makes them useful in the exploration of the lignocellulosic biomass for the production of fuel ethanol and other value-added commodity chemicals. In addition to this, endophytic fungi may become new sources of industrially useful enzymes such as lipases, amylases and proteases.
Assuntos
Ascomicetos/enzimologia , Endófitos/enzimologia , Proteínas Fúngicas/biossíntese , Amilases/biossíntese , Amilases/isolamento & purificação , Ascomicetos/genética , Ascomicetos/isolamento & purificação , Reatores Biológicos , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/isolamento & purificação , Endófitos/genética , Fermentação , Proteínas Fúngicas/isolamento & purificação , Peptídeo Hidrolases/biossíntese , Peptídeo Hidrolases/isolamento & purificaçãoRESUMO
Biological macromolecules are found in different shapes and sizes. Among these, enzymes catalyze biochemical reactions and are essential in all organisms, but is there a limit size for them to function properly? Large enzymes such as catalases have hundreds of kDa and are formed by multiple subunits, whereas most enzymes are smaller, with molecular weights of 20-60 kDa. Enzymes smaller than 10 kDa could be called microenzymes and the present literature review brings together evidence of their occurrence in nature. Additionally, bioactive peptides could be a natural source for novel microenzymes hidden in larger peptides and molecular downsizing could be useful to engineer artificial enzymes with low molecular weight improving their stability and heterologous expression. An integrative approach is crucial to discover and determine the amino acid sequences of novel microenzymes, together with their genomic identification and their biochemical biological and evolutionary functions.
Assuntos
Enzimas , Enzimas/química , Enzimas/genética , Enzimas/metabolismo , Humanos , Peso Molecular , Animais , Peptídeos/química , Peptídeos/metabolismoRESUMO
Aspergillus species have been highlighted in enzyme production looking for industrial applications, notably, amylases are one of the most interesting enzymes. They are capable of hydrolyzing α-glycosidic linkages of starch and widely used in industrial processes to produce ethanol, glucose, and fructose syrup as well as in the textiles, detergents, and paper industries applications. In this context, this work aimed at the biochemical characterization of the glucoamylase from Aspergillus japonicus and its application in the bio-bleaching process of recycled paper. The optimum temperature and pH for the glucoamylase assay were standardized as 50°C and 5.5. After 1 h of incubation, glucoamylase retained 90% of its activity at 30-50°C. It also kept 70% of its activity in the pH range of 4.0-6.5 after an hour of incubation. The enzyme led to an increase of 30% in the relative whiteness of 10 dry grams of sulfite paper and magazine paper when applied along with commercial cellulase and 10 mM MnCl2 . In addition, after the treatments, the glucoamylase recovered activity was 30%-32%, which indicates a prolonged availability of the enzyme and can considerably curtail the redundant downstream process of the recycled paper bio-bleaching. Thus, the glucoamylase from A. japonicus has a significant role in bio-bleaching recycled paper, reducing the necessity of hard chemicals, and improving the industrial process in an interesting economic and ecological mode.
Assuntos
Aspergillus , Glucana 1,4-alfa-Glucosidase , Glucana 1,4-alfa-Glucosidase/química , Temperatura , Amido , Concentração de Íons de HidrogênioRESUMO
Immobilization of enzymes on aminated supports using the glutaraldehyde chemistry may involve three different interactions, cationic, hydrophobic, and covalent interactions. To try to understand the impact this heterofunctionality, we study the physical adsorption of the beta-galactosidase from Aspergillus niger, on aminated supports (MANAE) and aminated supports with one (MANAE-GLU) or two molecules of glutaraldehyde (MANAE-GLU-GLU). To eliminate the chemical reactivity of the glutaraldehyde, the supports were reduced using sodium borohydride. After enzyme adsorption, the release of the enzyme from the supports using different NaCl concentrations, Triton X100, ionic detergents (SDS and CTAB), or different temperatures (4 °C to 55 °C) was studied. Using MANAE support, at 0.3 M NaCl almost all the immobilized enzyme was released. Using MANAE-GLU, 0.3 M, and 0.6 M NaCl similar results were obtained. However, incubation at 1 M or 2 M NaCl, many enzyme molecules were not released from the support. For the MANAE-GLU-GLU support, none of the tested concentrations of NaCl was sufficient to release all enzyme bound to the support. Only using high temperatures, 0.6 M NaCl, and 1 % CTAB or SDS, could the totality of the proteins be released from the support. The results shown in this paper confirm the heterofunctional character of aminated supports modified with glutaraldehyde.
Assuntos
Enzimas Imobilizadas , Cloreto de Sódio , Glutaral/química , Estabilidade Enzimática , Adsorção , Cetrimônio , Enzimas Imobilizadas/químicaRESUMO
The analysis of the secretome allows us to identify the proteins, especially carbohydrate-active enzymes (CAZymes), secreted by different microorganisms cultivated under different conditions. The CAZymes are divided into five classes containing different protein families. Thermothelomyces thermophilus is a thermophilic ascomycete, a source of many glycoside hydrolases and oxidative enzymes that aid in the breakdown of lignocellulosic materials. The secretome analysis of T. thermophilus LMBC 162 cultivated with submerged fermentation using tamarind seeds as a carbon source revealed 79 proteins distributed between the five diverse classes of CAZymes: 5.55% auxiliary activity (AAs); 2.58% carbohydrate esterases (CEs); 20.58% polysaccharide lyases (PLs); and 71.29% glycoside hydrolases (GHs). In the identified GH families, 54.97% are cellulolytic, 16.27% are hemicellulolytic, and 0.05 are classified as other. Furthermore, 48.74% of CAZymes have carbohydrate-binding modules (CBMs). Observing the relative abundance, it is possible to state that only thirteen proteins comprise 92.19% of the identified proteins secreted and are probably the main proteins responsible for the efficient degradation of the bulk of the biomass: cellulose, hemicellulose, and pectin.
RESUMO
In response to the escalating demand for sustainable agricultural methodologies, the utilization of microbial volatile organic compounds (VOCs) as antagonists against phytopathogens has emerged as a viable eco-friendly alternative. Microbial volatiles exhibit rapid diffusion rates, facilitating prompt chemical interactions. Moreover, microorganisms possess the capacity to emit volatiles constitutively, as well as in response to biological interactions and environmental stimuli. In addition to volatile compounds, these bacteria demonstrate the ability to produce soluble metabolites with antifungal properties, such as APE Vf, pyoverdin, and fragin. In this study, we identified two Pseudomonas strains (BJa3 and MCal1) capable of inhibiting the in vitro mycelial growth of the phytopathogenic fungus Aspergillus flavus, which serves as the causal agent of diseases in sugarcane and maize. Utilizing GC/MS analysis, we detected 47 distinct VOCs which were produced by these bacterial strains. Notably, certain volatile compounds, including 1-heptoxydecane and tridecan-2-one, emerged as primary candidates for inhibiting fungal growth. These compounds belong to essential chemical classes previously documented for their antifungal activity, while others represent novel molecules. Furthermore, examination via confocal microscopy unveiled significant morphological alterations, particularly in the cell wall, of mycelia exposed to VOCs emitted by both Pseudomonas species. These findings underscore the potential of the identified BJa3 and MCal1 Pseudomonas strains as promising agents for fungal biocontrol in agricultural crops.
RESUMO
Human population growth, industrialization, and globalization have caused several pressures on the planet's natural resources, culminating in the severe climate and environmental crisis which we are facing. Aiming to remedy and mitigate the impact of human activities on the environment, the use of lignocellulolytic enzymes for biofuel production, food, bioremediation, and other various industries, is presented as a more sustainable alternative. These enzymes are characterized as a group of enzymes capable of breaking down lignocellulosic biomass into its different monomer units, making it accessible for bioconversion into various products and applications in the most diverse industries. Among all the organisms that produce lignocellulolytic enzymes, microorganisms are seen as the primary sources for obtaining them. Therefore, this review proposes to discuss the fundamental aspects of the enzymes forming lignocellulolytic systems and the main microorganisms used to obtain them. In addition, different possible industrial applications for these enzymes will be discussed, as well as information about their production modes and considerations about recent advances and future perspectives in research in pursuit of expanding lignocellulolytic enzyme uses at an industrial scale.
RESUMO
The objective of this study was to evaluate the antioxidant activity, determine and quantify the phenolic compounds and other compounds, and evaluate the cellular cytotoxicity of mycelium extracts of two new Basidiomycete mushrooms strains isolated in Brazil and identified as Lepista sordida GMA-05 and Trametes hirsuta GMA-01. Higher amounts of proteins, free amino acids, total and reducing carbohydrates, and phenolic compounds as chlorogenic, ferulic, caffeic, and gallic acids were found in extracts of T. hirsuta and L. sordida. Protocatechuic acid was found only in aqueous extracts of L. sordida. The TLC of the extracts showed the predominance of glucose and smaller amounts of xylose. It was observed through UPLC-MS higher amounts of phenolic compounds. The aqueous extract from T. hirsuta had the most noteworthy results in the antioxidant assays, especially the ABTS test. The cytotoxic activity was evaluated using two different cell lineages and showed higher toxicity for L. sordida in macrophages J774-A1. However, in Vero cells, it was 12.6-fold less toxic when compared to T. hirsuta. Thus, both mushrooms show potential as functional foods or additives, presenting phenolic content, antioxidant activity, and low cytotoxic activity in the tested cells.
Assuntos
Agaricales , Trametes , Animais , Antioxidantes/análise , Antioxidantes/farmacologia , Brasil , Chlorocebus aethiops , Cromatografia Líquida , Micélio/química , Extratos Vegetais/química , Polyporaceae , Espectrometria de Massas em Tandem , Trametes/química , Células VeroRESUMO
Fungi producing high xylanase levels have attracted considerable attention because of their potential industrial applications. Batch cultivations of Aspergillus terricola fungus were evaluated in stirred tank and airlift bioreactors, by using wheat bran particles suspended in the cultivation medium as substrate for xylanase and ß-xylosidase production. In the stirred tank bioreactor, in physical conditions of 30°C, 300 rpm, and aeration of 1 vvm (1 l min⻹), with direct inoculation of fungal spores, 7,475 U l⻹ xylanase was obtained after 36 h of operation, remaining constant after 24 h. In the absence of air injection in the stirred tank reactor, limited xylanase production was observed (final concentration 740 U l⻹). When the fermentation process was realized in the airlift bioreactor, xylanase production was higher than that observed in the stirred tank bioreactor, being 9,265 U l⻹ at 0.07 vvm (0.4 l min⻹) and 12,845 U l⻹ at 0.17 vvm (1 l min⻹) aeration rate.
Assuntos
Aspergillus/enzimologia , Reatores Biológicos/microbiologia , Xilosidases/metabolismo , Aspergillus/metabolismo , Fibras na Dieta , FermentaçãoRESUMO
Solid-state fermentation obtained from different and low-cost carbon sources was evaluated to endocellulases and endoxylanases production by Aspergillus japonicus C03. Regarding the enzymatic production the highest levels were observed at 30 °C, using soy bran added to crushed corncob or wheat bran added to sugarcane bagasse, humidified with salt solutions, and incubated for 3 days (xylanase) or 6 days (cellulase) with 70% relative humidity. Peptone improved the xylanase and cellulase activities in 12 and 29%, respectively. The optimum temperature corresponded to 60 °C and 50-55 °C for xylanase and cellulase, respectively, both having 4.0 as optimum pH. Xylanase was fully stable up to 40 °C, which is close to the rumen temperature. The enzymes were stable in pH 4.0-7.0. Cu++ and Mn++ increased xylanase and cellulase activities by 10 and 64%, respectively. A. japonicus C03 xylanase was greatly stable in goat rumen fluid for 4 h during in vivo and in vitro experiments.
Assuntos
Aspergillus/enzimologia , Celulase/biossíntese , Celulase/química , Endo-1,4-beta-Xilanases/biossíntese , Endo-1,4-beta-Xilanases/química , Aditivos Alimentares , Ração Animal , Animais , Aspergillus/metabolismo , Carbono/metabolismo , Celulase/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Fermentação , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Cabras , Concentração de Íons de Hidrogênio , Microbiologia Industrial , Resíduos Industriais/análise , Nitrogênio/metabolismo , Nitrogênio/provisão & distribuição , Rúmen/enzimologia , Rúmen/metabolismo , Ruminantes , TemperaturaRESUMO
Interest in chitin-degrading enzymes has grown over the years, and microbial chitinases are the most attractive and promising candidates for the control of plant pests (fungi and insects). Currently, there are many studies on chitinases produced by cultivable microorganisms; however, almost none of them have achieved acceptable applicability as a biopesticide in the field. Approximately 99% of the microorganisms from soil cannot be isolated by conventional culture-dependent methods, thus having an enormous biotechnological/genetic potential to be explored. On the basis of this, the present paper aims to provide a brief overview of the metagenomic opportunities that have been emerging and allowing access to the biochemical potential of uncultivable microorganisms through the direct mining of DNA sequences recovered from the environment. This work also shortly discussed the future perspectives of functional and sequence-based metagenomic approaches for the identification of new chitinase-coding genes with potential for applications in several agricultural and biotechnological industries, especially in biological control.
Assuntos
Quitinases , Animais , Agentes de Controle Biológico , Quitina , Quitinases/genética , Fungos/genética , MetagenômicaRESUMO
A purified exo-polygalacturonase of Neosartorya glabra (EplNg) was successfully characterized. EplNg native presented 68.2 kDa, with 32% carbohydrate content. The deglycosylated form showed 46.3 kDa and isoelectric point of 5.4. The identity of EplNg was confirmed as an exo-polygalacturonase class I (EC 3.2.1.67) using mass spectrometry and Western-Blotting. Capillary electrophoresis indicated that only galacturonic acid was released by the action of EplNg on sodium polypectate, confirming an exoenzyme character. The structural model confers that EplNg has a core formed by twisted parallel ß-sheets structure. Among twelve putative cysteines, ten were predicted to form disulfide bridges. The catalytic triad predicted is composed of Asp223, Asp245, and Asp246 aligned along with a distance in 4-5 Å, suggesting that EplNg probably does not perform the standard inverting catalytic mechanism described for the GH28 family. EplNg was active from 30 to 90 °C, with maximum activity at 65 °C, pH 5.0. The Km and Vmax determined using sodium polypectate were 6.9 mg·mL-1 and Vmax 690 µmol·min-1.mg-1, respectively. EplNg was active and stable over a wide range of pH values and temperatures, confirming the interesting properties EplNg and provide a basis for the development of the enzyme in different biotechnological processes.
Assuntos
Aspergillus/enzimologia , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/metabolismo , Catálise , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificação , Ácidos Hexurônicos/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Pectinas/metabolismo , Conformação Proteica , Estabilidade Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , TemperaturaRESUMO
The lignocellulosic biomass comprises three main components: cellulose, hemicellulose, and lignin. Degradation and conversion of these three components are attractive to biotechnology. This study aimed to prospect fungal lignocellulolytic enzymes with potential industrial applications, produced through a temporal analysis using Hymenaea courbaril and Tamarindus indica seeds as carbon sources. α-L-arabinofuranosidase, acetyl xylan esterase, endo-1,5-α-L-arabinanase, ß-D-galactosidase, ß-D-glucosidase, ß-glucanase, ß-D-xylosidase, cellobiohydrolase, endoglucanase, lichenase, mannanase, polygalacturonase, endo-1,4-ß-xylanase, and xyloglucanase activities were determined. The enzymes were produced for eight filamentous fungi: Aspergillus fumigatus, Trametes hirsuta, Lasiodiplodia sp., two strains of Trichoderma longibrachiatum, Neocosmospora perseae, Fusarium sp. and Thermothelomyces thermophilus. The best producers concerning enzymatic activity were T. thermophilus and T. longibrachiatum. The optimal conditions for enzyme production were the media supplemented with tamarind seeds, under agitation, for 72 h. This analysis was essential to demonstrate that cultivation conditions, static and under agitation, exert strong influences on the production of several enzymes produced by different fungi. The kind of sugarcane, pretreatment used, microorganisms, and carbon sources proved limiting sugar profile factors.
RESUMO
Treatment of Aspergillus niveus with 30 µg tunicamycin/ml did not interfere with α-glucosidase production, secretion, or its catalytic properties. Fully- and under-glycosylated forms of the enzyme had similar molecular masses, ~56 kDa. Moreover, the absence of N-glycans did not affect either pH optimum (6.0) or temperature optimum (65°C). The K(m) and V(max) values of under- and fully-glycosylated forms of α-glucosidase were similar when assessed for hydrolysis of starch (~0.6 mg/ml, ~350 µmol glucose per min per ml), maltose (~0.54 µmol, ~330 µmol glucose per min per ml) and p-nitrophenyl-α-D: -glucopyranoside (~0.54 µmol, ~8.28 µmol p-nitrophenol per min per ml). However, the under-glycosylated form was sensitive to high temperatures probably because, in addition to stabilizing the protein conformation, glycosylation may also prevent unfolded or partially folded proteins from aggregating. Binding assays clearly showed that the under-glycosylated protein did not bind to concanavalin A but has conserve its jacalin-binding property, suggesting that only O-glycans might be intact on the tunicamycin treated form of the enzyme.
Assuntos
Aspergillus/efeitos dos fármacos , Aspergillus/enzimologia , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Inibidores de Glicosídeo Hidrolases , Tunicamicina/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Glicosilação/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Cinética , Maltose/metabolismo , Peso Molecular , Nitrofenilgalactosídeos/metabolismo , Amido/metabolismo , Temperatura , alfa-Glucosidases/química , alfa-Glucosidases/isolamento & purificação , alfa-Glucosidases/metabolismoRESUMO
Today, many microbial amylases are available commercially and they have almost completely replaced chemical hydrolysis in several industry processes. Amylases from microorganisms have a broad spectrum of industrial applications as they are more stable than amylases obtained from plants and animals. The objective of this work was to use potato baits in an Atlantic Forest remnant located in Ribeirão Preto, São Paulo, Brazil, in order to obtain amylase-producing fungi with potential for biotechnological application. In addition, the culture conditions for the fungal strain that presented higher production of glucoamylase were standardized using industrial wastes. For this, 6 PET bottles containing potatoes as baits were scattered at different points in an Atlantic forest remnant. After 6 days, the samples were collected, and the filamentous fungi were isolated in Petri dishes. Fungi screening was carried out in Khanna liquid medium with 1% starch Reagen®, at 30 °C, pH 6.0, under static conditions for 4 days. Proteins and glucoamylase activity were determined by Bradford and 3,5-dinitrosalicylic acid (DNS), respectively. Among all isolated fungi, A. carbonarius showed the highest glucoamylase production. Its best cultivation conditions were observed in Khanna medium, 4 days, at 30 °C, pH 6.0, under static condition with 0.1% yeast extract and 1% starch Reagen®. Wheat and brewing residues were also used as inducers for large quantities of glucoamylase production. A. carbonarius showed to be a good alternative for the wheat and brewing waste destinations in order to obtain high added value products.
Assuntos
Aspergillus/enzimologia , Aspergillus/isolamento & purificação , Glucana 1,4-alfa-Glucosidase/metabolismo , Triticum/metabolismo , Bioprospecção , Brasil , Florestas , Hidrólise , Amido/metabolismo , Clima TropicalRESUMO
The use of non-potable water (such as seawater) is an attractive alternative for water intensive processes such as biomass pretreatment and saccharification steps in the production of biochemicals and biofuels. Identification and application of halotolerant enzymes compatible with high-salt conditions may reduce the energy needed for non-potable water treatment and decrease waste treatment costs. Here we present the biochemical properties of a halotolerant endo-1,4-ß-xylanase produced by Aspergillus clavatus in submerged fermentation, using paper sludge (XPS) and sugarcane bagasse (XSCB), and its potential application in the hydrolysis of agroindustrial residues. The peptide mass fingerprint and amino acid sequencing of the XPS and XSCB enzymes showed primary structure similarities with an endo-1,4-ß-xylanase from Aspergillus clavatus (XYNA_ASPCL). Both enzyme preparations presented good thermal stability at 50 °C and were stable over a wide range of pH and Vmax up to 2450 U/mg for XPS. XPS and XSCB were almost fully stable even after 24 h of incubation in the presence of up to 3 M NaCl, and their activity were not affected by 500 mM NaCl. Both enzyme preparations were capable of hydrolyzing paper sludge and sugarcane bagasse to release reducing sugars. These characteristics make this xylanase attractive to be used in the hydrolysis of biomass, particularly with brackish water or seawater.