Thermothelomyces thermophilus cultivated with residues from the fruit pulp industry: enzyme immobilization on ionic supports of a crude cocktail with enhanced production of lichenase.

ß-Glucans comprise a group of ß-D-glucose polysaccharides (glucans) that occur naturally in the cell walls of bacteria, fungi, and cereals. Its degradation is catalyzed by ß-glucanases, enzymes that catalyze the breakdown of ß-glucan into cello-oligosaccharides and glucose. These enzymes are classified as endo-glucanases, exo-glucanases, and glucosidases according to their mechanism of action, being the lichenases (ß-1,3;1,4-glucanases, EC 3.2.1.73) one of them. Hence, we aimed to enhance lichenase production by Thermothelomyces thermophilus through the application of response surface methodology, using tamarind (Tamarindus indica) and jatoba (Hymenaea courbaril) seeds as carbon sources. The crude extract was immobilized, with a focus on improving lichenase activity, using various ionic supports, including MANAE (monoamine-N-aminoethyl), DEAE (diethylaminoethyl)-cellulose, CM (carboxymethyl)-cellulose, and PEI (polyethyleneimine)-agarose. Regarding lichenase, the optimal conditions yielding the highest activity were determined as 1.5% tamarind seeds, cultivation at 50 °C under static conditions for 72 h. Moreover, transitioning from Erlenmeyer flasks to a bioreactor proved pivotal, resulting in a 2.21-fold increase in activity. Biochemical characterization revealed an optimum temperature of 50 °C and pH of 6.5. However, sustained stability at varying pH and temperature levels was challenging, underscoring the necessity of immobilizing lichenase on ionic supports. Notably, CM-cellulose emerged as the most effective immobilization medium, exhibiting an activity of 1.01 U/g of the derivative (enzyme plus support), marking a substantial enhancement. This study marks the first lichenase immobilization on these chemical supports in existing literature.

Novel Pseudomonas Species Prevent the Growth of the Phytopathogenic Fungus Aspergillus flavus.

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.

Microenzymes: Is There Anybody Out There?

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.

Secretome Analysis of Thermothelomyces thermophilus LMBC 162 Cultivated with Tamarindus indica Seeds Reveals CAZymes for Degradation of Lignocellulosic Biomass.

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.

Adsorption features of reduced aminated supports modified with glutaraldehyde: Understanding the heterofunctional features of these supports.

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.

Biochemical characterization of an acid-thermostable glucoamylase from Aspergillus japonicus with potential application in the paper bio-deinking.

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.

Trichoderma longibrachiatum and thermothelomyces thermophilus co-culture: improvement the saccharification profile of different sugarcane bagasse varieties.

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.

Lignocellulolytic Biocatalysts: The Main Players Involved in Multiple Biotechnological Processes for Biomass Valorization.

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.

Biochemical characterization and biological properties of mycelium extracts from Lepista sordida GMA-05 and Trametes hirsuta GMA-01: new mushroom strains isolated in Brazil.

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.

Structural model and functional properties of an exo-polygalacturonase from Neosartorya glabra.

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.

Prospection of Fungal Lignocellulolytic Enzymes Produced from Jatoba (Hymenaea courbaril) and Tamarind (Tamarindus indica) Seeds: Scaling for Bioreactor and Saccharification Profile of Sugarcane Bagasse.

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.

Perspectives on Expanding the Repertoire of Novel Microbial Chitinases for Biological Control.

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.

A Highly Glucose Tolerant ß-Glucosidase from Malbranchea pulchella (MpBg3) Enables Cellulose Saccharification.

ß-glucosidases catalyze the hydrolysis ß-1,4, ß-1,3 and ß-1,6 glucosidic linkages from non-reducing end of short chain oligosaccharides, alkyl and aryl ß-D-glucosides and disaccharides. They catalyze the rate-limiting reaction in the conversion of cellobiose to glucose in the saccharification of cellulose for second-generation ethanol production, and due to this important role the search for glucose tolerant enzymes is of biochemical and biotechnological importance. In this study we characterize a family 3 glycosyl hydrolase (GH3) ß-glucosidase (Bgl) produced by Malbranchea pulchella (MpBgl3) grown on cellobiose as the sole carbon source. Kinetic characterization revealed that the MpBgl3 was highly tolerant to glucose, which is in contrast to many Bgls that are completely inhibited by glucose. A 3D model of MpBgl3 was generated by molecular modeling and used for the evaluation of structural differences with a Bgl3 that is inhibited by glucose. Taken together, our results provide new clues to understand the glucose tolerance in GH3 ß-glucosidases.

Fungal communities differentially respond to warming and drought in tropical grassland soil.

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.

A Halotolerant Endo-1,4-ß-Xylanase from Aspergillus clavatus with Potential Application for Agroindustrial Residues Saccharification.

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.

Novel amylase-producing fungus hydrolyzing wheat and brewing residues, Aspergillus carbonarius, discovered in tropical forest remnant.

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.

Efficient hydrolysis of wine and grape juice anthocyanins by Malbranchea pulchella ß-glucosidase immobilized on MANAE-agarose and ConA-Sepharose supports.

ß-glucosidases (BGLs) hydrolyze short-chain cellulooligosaccharides. Some BGLs can hydrolyze anthocyanins and be applied in the clarification process of food industries, especially grape juice and wine. Enzyme immobilization is a valuable tool to increase enzyme stabilization. In this work, Malbranchea pulchella BGL was immobilized on Monoaminoethyl-N-ethyl-agarose ionic support, MANAE-agarose, and Concanavalin A-Sepharose affinity support, Con-A-Sepharose. The formed biocatalysts, denominated BLG-MANAE and BLG-ConA, were applied in the grape juice and red wine clarification. BGL-MANAE and BGL-ConA hyperactivated M. pulchella BGL 10- and 3-fold, respectively. Both biocatalysts showed at least 70% activity at pH range 2-11, until 24 h incubation. BGL-MANAE and BGL-ConA showed activity of 60% and 100%, respectively, at 50 °C, up to 24 h. Both biocatalysts were efficiently reused 20-fold. They were stable in the presence of up to 0.1 M glucose for 24 h incubation, and with 5%, 10% and 15% ethanol kept up to 70% activity. BGL-MANAE biocatalyst was 11% and 25% more efficient than BGL-ConA in clarification of concentrate and diluted wines, respectively. Likewise, BGL-MANAE biocatalysts were 14% and 33% more efficient than the BGL-ConA in clarification of diluted and concentrated juices, respectively. Therefore, the BGL-MANAE biocatalyst was especially effective in red wine and grape juice clarification.