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.

Purification and functional properties of a novel glucoamylase activated by manganese and lead produced by Aspergillus japonicus.

Microbial amylases are used to produce ethanol, glucose and can be applied in textiles products, detergents and other industries. This study aimed to determine the best carbon source concentration to induce the amylase production by A. japonicus, and its purification and biochemical characterization. For that, this fungus was cultivated in Khanna medium, pH 5.5, for 4 days, at 25°C, in static condition, supplemented with potato starch and maltose in different concentrations. The fungal crude enzymatic extract was purified in a unique elution in DEAE-cellulose column and the molecular mass was determined as 72kDa. The optimum temperature and pH was 65°C and 5.0, respectively. Amylase remained 75% of its activity after one hour at 50°C and was stable in the pH range 3.0-7.0. The analysis of the end-products by thin layer chromatography showed only glucose formation, which characterizes the purified enzyme as a glucoamylase. Amylopectin was the best substrate for the enzyme assay and Mn+2 and Pb+2 were good glucoamylase activators. This activation, in addition to the biochemical characteristics are important results for future biotechnological applications of this glucoamylase in the recycling and deinking process by the paper industries.