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.

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.

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.

The functional properties of a xyloglucanase (GH12) of Aspergillus terreus expressed in Aspergillus nidulans may increase performance of biomass degradation.

Filamentous fungi are attractive hosts for heterologous protein expression due to their capacity to secrete large amounts of enzymes into the extracellular medium. Xyloglucanases, which specifically hydrolyze xyloglucan, have been recently applied in lignocellulosic biomass degradation and conversion in many other industrial processes. In this context, this work aimed to clone, express, and determine the functional properties of a recombinant xyloglucanase (AtXEG12) from Aspergillus terreus, and also its solid-state (SSF) and submerged (SmF) fermentation in bioreactors. The purified AtXEG12 showed optimum pH and temperature of 5.5 and 65 °C, respectively, demonstrating to be 90 % stable after 24 h of incubation at 50 °C. AtXEG12 activity increased in the presence of 2-mercaptoethanol (65 %) and Zn+2 (45 %), while Cu+2 and Ag+ ions drastically decreased its activity. A substrate assay showed, for the first time for this enzyme's family, xylanase activity. The enzyme exhibited high specificity for tamarind xyloglucan (K M 1.2 mg mL-1) and V max of 17.4 µmol min-1 mg-1 of protein. The capillary zone electrophoresis analysis revealed that AtXEG12 is an endo-xyloglucanase. The heterologous xyloglucanase secretion was greater than the production by wild-type A. terreus cultivated in SmF. On the other hand, AtXEG12 activity reached by SSF was sevenfold higher than values achieved by SmF, showing that the expression of recombinant enzymes can be significantly improved by cultivation under SSF.

Screening of thermotolerant and thermophilic fungi aiming ß-xylosidase and arabinanase production.

Plant cell wall is mainly composed by cellulose, hemicellulose and lignin. The heterogeneous structure and composition of the hemicellulose are key impediments to its depolymerization and subsequent use in fermentation processes. Thus, this study aimed to perform a screening of thermophilic and thermotolerant filamentous fungi collected from different regions of the São Paulo state, and analyze the production of ß-xylosidase and arabinanase at different temperatures. These enzymes are important to cell wall degradation and synthesis of end products as xylose and arabinose, respectively, which are significant sugars to fermentation and ethanol production. A total of 12 fungal species were analyzed and 9 of them grew at 45 °C, suggesting a thermophilic or thermotolerant character. Additionally Aspergillus thermomutatus anamorph of Neosartorya and A. parasiticus grew at 50 °C. Aspergillus niger and Aspergillus thermomutatus were the filamentous fungi with the most expressive production of ß-xylosidase and arabinanase, respectively. In general for most of the tested microorganisms, ß-xylosidase and arabinanase activities from mycelial extract (intracellular form) were higher in cultures grown at high temperatures (35-40 °C), while the correspondent extracellular activities were favorably secreted from cultures at 30 °C. This study contributes to catalogue isolated fungi of the state of São Paulo, and these findings could be promising sources for thermophilic and thermotolerant microorganisms, which are industrially important due to their enzymes.