Biochemical Characterization of a Novel Thermostable 1,4-α-Glucoamylase from Aspergillus brasiliensis Strain Isolated in the Brazilian Atlantic Forest.

Glucoamylases are exo-enzymes that cleave the ends of the starch chain, releasing glucose units. In the current work, we described a novel 1,4-α-glucoamylase from an A. brasiliensis strain isolated from an environmental sample. The purified glucoamylase, GlaAb, has a molecular mass of 69 kDa and showed a starch binding domain. GlaAb showed a similar sequence to other fungal glucoamylases, and the molecular 3D model analysis of GlaAb suggests an overall structure as described in the literature, except by elongation in the loop connecting the 4th and 5th α-helices. The enzyme showed activity over a wide range of pH and temperature, with maximum activity at pH 4.5 and 60 °C. GlaAb was stable at 50 °C for 7 h, maintaining 67% residual activity, and it was not inhibited by glucose up to 0.1 M. The glucoamylase was 65% more active in the presence of Mn2+ and showed a Km of 2.21 mg mL-1, Vmax of 155 U mg-1, Kcat 179 s-1, and Kcat/Km 81.06 mg mL-1 s-1 using potato starch as substrate. The results obtained are promising and provide the basis for the development of applications of GlaAb in the industrial process.

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.

Increased Malbranchea pulchella ß-glucosidase production and its application in agroindustrial residue hydrolysis: A research based on experimental designs.

ß-Glucosidases are a limiting factor in the conversion of cellulose to glucose for the subsequent ethanol production. Here, ß-glucosidase production by Malbranchea pulchella was optimized using Composite Central Designs and Response Surface Methodologies from a medium designed. The coefficient of determination (R2 ) was 0.9960, F-value was very high, and the lack of fit was found to be non-significant. This indicates a statistic valid and predictive result. M. pulchella enzymatic extract was successfully tested as an enzymatic cocktail in a mixture design using sugarcane bagasse, soybean hull and barley bagasse. We proved that the optimization of the ß-glucosidase production and the application in hydrolysis using unexpansive biomass and agricultural wastes can be accomplished by means of statistical methodologies. The strategy presented here can be useful for the improvement of enzyme production and the hydrolysis process, arising as an alternative for bioeconomy.

Trametes versicolor laccase production using agricultural wastes: a comparative study in Erlenmeyer flasks, bioreactor and tray.

Laccases are very interesting biocatalysts of recognized importance for several industrial applications. Its production by Trametes versicolor, a white-rot fungus, was induced by a combination of cotton gin wastes (1%), a lignocellulosic waste, and vinasse (15%), an industrial by-product from sugarcane industry. The use of these agro-industrial wastes are interesting, since it helps in reducing the enzyme production costs, due to their low cost and wide availability, as well as the environmental contamination issues, due to their improper disposal. Thus, laccase production was studied in submerged fermentation of T. versicolor using these agro-industrial wastes (cotton gin waste and vinasse) as carbon source and an additional nitrogen source (0.1% peptone). Three different bioreactors were evaluated for laccase production, such as BioFlo 310 bioreactor, aluminium tray and Erlenmeyer flasks to achieve high levels of laccase production. The highest specific production of laccase was found in BioFlo 310 bioreactor with 12 days of fermentation (55.24 U/mg prot.), which has been shown to be closely related to the oxygen supply to the microorganism through aeration of the fermentation medium. This study brings new insights into green biotechnology regarding vinasse utilization, which is frequently discharged in soils, rivers, and lakes causing adverse effects on agricultural soils and biota, as well as the cotton gin waste recovery.