Prospection of Psychrotrophic Filamentous Fungi Isolated from the High Andean Paramo Region of Northern Ecuador: Enzymatic Activity and Molecular Identification.

The isolation of filamentous fungal strains from remote habitats with extreme climatic conditions has led to the discovery of a series of enzymes with attractive properties that can be useful in various industrial applications. Among these, cold-adapted enzymes from fungi with psychrotrophic lifestyles are valuable agents in industrial processes aiming towards energy reduction. Out of eight strains isolated from soil of the paramo highlands of Ecuador, three were selected for further experimentation and identified as Cladosporium michoacanense, Cladosporium sp. (cladosporioides complex), and Didymella sp., this last being reported for the first time in this area. The secretion of seven enzymes, namely, endoglucanase, exoglucanase, ß-D-glucosidase, endo-1,4-ß-xylanase, ß-D-xylosidase, acid, and alkaline phosphatases, were analyzed under agitation and static conditions optimized for the growth period and incubation temperature. Cladosporium strains under agitation as well as incubation for 72 h mostly showed the substantial activation for endoglucanase reaching up to 4563 mU/mL and xylanase up to 3036 mU/mL. Meanwhile, other enzymatic levels varied enormously depending on growth and temperature. Didymella sp. showed the most robust activation at 8 °C for endoglucanase, ß-D-glucosidase, and xylanase, indicating an interesting profile for applications such as bioremediation and wastewater treatment processes under cold climatic conditions.

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.

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.

Yeasts in the nests of the leaf-cutter ant Acromyrmex balzani in a Savanna biome: exploitation of community and metabolic diversity.

The leaf-cutter ant Acromyrmex balzani is responsible for causing important losses in reforestation areas, crops, and pastures, and is frequently found in the Brazilian savanna (Cerrado). So far, there is no information regarding the yeast communities that occur in their nests. Here, we evaluated the diversity, composition, and structure of yeast communities in both fungus gardens (FG) and external refuse dump (RD) of this ant species (Palmas, Tocantins, northern Brazil). A total of 720 yeasts were isolated, comprising 52 species distributed in 29 genera. The RDs have significantly richer and more diverse yeast communities than the fungus gardens, regardless of the season and the level of preservation in the area. The isolates produced a wide range of carbon polymer-degrading enzymes and were able to assimilate carbon-sources present in plant materials. We observed a different proportion of enzyme-producers and carbon-assimilation found in external refuse dump and fungus gardens from preserved and disturbed areas, suggesting that this interaction may vary depending on the environmental conditions. A. balzani nests in the savanna biome are a hotspot of yeast species with ecological, clinical, and biotechnological implications.

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.

Genome mining for peptidases in heat-tolerant and mesophilic fungi and putative adaptations for thermostability.

BACKGROUND: Peptidases (EC 3.4) consist of a large group of hydrolytic enzymes that catalyze the hydrolysis of proteins accounting for approximately 65% of the total worldwide enzyme production. Peptidases from thermophilic fungi have adaptations to high temperature that makes them adequate for biotechnological application. In the present study, we profiled the genomes of heat-tolerant fungi and phylogenetically related mesophilic species for genes encoding for peptidases and their putative adaptations for thermostability. RESULTS: We generated an extensive catalogue of these enzymes ranging from 241 to 820 peptidase genes in the genomes of 23 fungi. Thermophilic species presented the smallest number of peptidases encoding genes in relation to mesophilic species, and the peptidases families with a greater number of genes were the most affected. We observed differences in peptidases in thermophilic species in comparison to mesophilic counterparts, at (i) the genome level: a great reduction in the number of peptidases encoding genes that harbored a higher number of copies; (ii) in the primary protein structure: shifts in proportion of single or groups of amino acids; and (iii) in the three-dimensional structure: reduction in the number of internal cavities. Similar results were reported for extremely thermophilic proteins, but here we show for the first time that several changes also occurred on the moderate thermophilic enzymes of fungi. In regards to the amino acids composition, peptidases from thermophilic species in relation to the mesophilic ones, contained a larger proportion of Ala, Glu, Gly, Pro, Arg and Val residues and a lower number of Cys, His, Ile, Lys, Met, Asn, Gln, Ser, Thr and Trp residues (P < 0.05). Moreover, we observed an increase in the proportion of hydrophobic and charged amino acids and a decrease in polar amino acids. CONCLUSIONS: Although thermophilic fungi present less genes encoding for peptidases, these have adaptations that could play a role in thermal resistance from genome to protein structure level.

Biochemical properties and evaluation of washing performance in commercial detergent compatibility of two collagenolytic serine peptidases secreted by Aspergillus fischeri and Penicillium citrinum.

Filamentous fungi secrete diverse peptidases with different biochemical properties, which is of considerable importance for application in various commercial sectors. In this study, we describe the isolation of two fungal species collected from the soil of decayed organic matter: Aspergillus fischeri and Penicillium citrinum. In a submerged bioprocess, we observed better peptidase production with the fungus P. citrinum, which reached a peak production at 168 h with 760 U/mL, in comparison with the fungus A. fischeri, which reached a peak production at 72 h with 460 U/mL. In both situations, the fermentative medium contained 0.5% crushed feathers as a source of nitrogen. On performing biochemical characterization, we detected two alkaline serine peptidases: The one secreted by P. citrinum had optimal activity at pH 7.0 and at 45°C, while the one secreted by A. fischeri had optimal activity in pH 6.5-8 and at 55-60°C. Metallic ions were effective in modulating these peptidases; in particular, Cu2+ promoted negative modulation of both peptidases. The peptidases were stable and functional under conditions of nonionic surfactants, temperatures up to 45°C for 1 h, and incubation over a wide pH range. In addition, it was observed that both peptidases had the capacity to hydrolyze collagen and performed well in removing an egg protein stain when supplemented into a commercial powder detergent; this was especially true for the peptidase from P. citrinum.

Evaluation of the catalytic specificity, biochemical properties, and milk clotting abilities of an aspartic peptidase from Rhizomucor miehei.

In this study, we detail the specificity of an aspartic peptidase from Rhizomucor miehei and evaluate the effects of this peptidase on clotting milk using the peptide sequence of k-casein (Abz-LSFMAIQ-EDDnp) and milk powder. Molecular mass of the peptidase was estimated at 37 kDa, and optimum activity was achieved at pH 5.5 and 55 °C. The peptidase was stable at pH values ranging from 3 to 5 and temperatures of up 45 °C for 60 min. Dramatic reductions in proteolytic activity were observed with exposure to sodium dodecyl sulfate, and aluminum and copper (II) chloride. Peptidase was inhibited by pepstatin A, and mass spectrometry analysis identified four peptide fragments (TWSISYGDGSSASGILAK, ASNGGGGEYIFGGYDSTK, GSLTTVPIDNSR, and GWWGITVDRA), similar to rhizopuspepsin. The analysis of catalytic specificity showed that the coagulant activity of the peptidase was higher than the proteolytic activity and that there was a preference for aromatic, basic, and nonpolar amino acids, particularly methionine, with specific cleavage of the peptide bond between phenylalanine and methionine. Thus, this peptidase may function as an important alternative enzyme in milk clotting during the preparation of cheese.

Thermophilic fungi in the new age of fungal taxonomy.

Thermophilic fungi are of wide interest due to their potential to produce heat-tolerant enzymes for biotechnological processes. However, the taxonomy of such organisms remains obscure, especially given new developments in the nomenclature of fungi. Here, we examine the taxonomy of the thermophilic fungi most commonly used in industry in light of the recent taxonomic changes following the adoption of the International Code of Nomenclature for Algae, Fungi and Plants and also based on the movement One Fungus = One Name. Despite the widespread use of these fungi in applied research, several thermotolerant fungi still remain classified as thermophiles. Furthermore, we found that while some thermophilic fungi have had their genomes sequenced, many taxa still do not have barcode sequences of reference strains available in public databases. This lack of basic information is a limiting factor for the species identification of thermophilic fungi and for metagenomic studies in this field. Based on next-generation sequencing, such studies generate large amounts of data, which may reveal new species of thermophilic fungi in different substrates (composting systems, geothermal areas, piles of plant material). As discussed in this study, there are intrinsic problems associated with this method, considering the actual state of the taxonomy of thermophilic fungi. To overcome such difficulties, the taxonomic classification of this group should move towards standardizing the commonly used species names in industry and to assess the possibility of including new systems for describing species based on environmental sequences.